The Karasuma Group (烏丸グループ Karasuma Gurūpu?), alias the Black Organization (黒の組織 Kuro no Soshiki?), serves as the main antagonist in the series. It is a secret criminal syndicate with a hidden objective. To achieve that goal, the Black Organization commits various crimes to maintain its secrecy, remove obstacles, and gather funding and resources for its mysterious research projects. The members of the organization are given code names based on alcoholic beverages. Their boss is Renya Karasuma and the first members to appear were Gin and Vodka, who were responsible for transforming Shinichi Kudo into a child with the enigmatic drug APTX 4869
- Renya Karasuma: The primary antagonist of the series. Leader of the Black Organization, he is respectfully referred to as "that person" (ano kata) by lower ranked members of the Black Organization.
- Rum: The second main antagonist of the series. Second in charge of the Black Organization, his physical description varies: an elderly man, a feminine man, and a strongly-built man; but all sources agree that he has an artificial eye. The three main suspects are Hyoue Kuroda, Rumi Wakasa, and Kanenori Wakita
- Gin: A high ranking Black Organization executive official who seems to specialize in coordinating deals and assassinations. He is one of the most frequently recurring Black Organization characters. He is shown to be highly intelligent, lethal, and very acute. His long silver hair and sweeping trench coat give him a distinctive appearance. He forced the drug APTX 4869 on Shinichi.
- Vermouth (Sharon Vineyard/Chris Vineyard): A mysterious actress from America with exceptional disguise and voice changing abilities that allow her to pose as anyone. Like Gin, she is a Black Organization executive member. She is hated by a number of the members of the Black Organization, but due to the fact that she is their boss' favorite, she is untouchable and they are unable to do anything to her. It is unknown where her loyalty lies, as she often protects Conan (whom she knows to be Shinichi) and calls him a "silver bullet", implying that she believes him to be capable of bringing down the Black Organization. She also does not seem to age.
- Vodka: Gin's secretary and assistant, he is another frequently recurring Black Organization character. He wears a hat and sunglasses at all times. He is less intelligent compared to his boss Gin whom he often is seen working with.
- Chianti: A professional sniper who has been seen to take orders from Gin. She has a fiery and impatient personality. She loathes Vermouth.
- Korn: A professional sniper who is Chianti's partner and who has been seen to take orders from Gin. His speech is characterized by short phrases with many pauses.
Spies for other organizations
- Kir: Operates under alias of Rena Mizunashi, a TV reporter. Her true name is Hidemi Hondou. Like her deceased father, she is actually a member of the CIA who infiltrated the Black Organization. Her brother, Eisuke Hondou, was not aware of this since Hidemi pretended not to know him after she went undercover.
- Bourbon: An operative specializing in deduction and intelligence gathering skills. He was initially dispatched to look for Sherry, but spent more of that time investigating Shuichi Akai whom he hates for killing his partner Scotch. He works for the Japanese Secret Police. His true name is Rei Furuya, but he goes by Tooru Amuro in public.
- Tequila: An agent with the same rank as Vodka who seemed to primarily deal with programmers. He was a giant man who spoke with an Osakan accent. He was unintentionally killed in explosion.
- Pisco: An elderly executive agent who was also the chairman of a car company. He was killed by Gin after being caught on camera performing an assassination. He discovered Haibara was Sherry, but was unable to inform anyone before he was killed by Gin.
- Calvados: A sniper infatuated with Vermouth who shot himself after being crippled by Shuichi Akai.
- Akemi Miyano: Shiho Miyano's older sister and Shuichi Akai's girlfriend. She was a low ranking member of the organization and completed a billion yen robbery as part of an agreement to release herself and her sister from the organization. The organization reneged on the deal and she was killed by Gin.
- Atsushi Miyano: Haibara's father and Elena's husband. He headed the APTX 4869 project before Shiho Miyano took over. Both he and Elena died for some reason which Pisco called an accident while Shiho was young.
- Elena Miyano: Haibara's mother and Atsushi's wife. She was of British origin and was known within the organization as "Hell's Angel". She worked the APTX 4869 project before Shiho Miyano took over. Both she and Atsushi died for some reason which Pisco called an accident while Shiho was young. She left some mysterious tapes for Shiho.
- Rikumichi Kusuda: An agent sent to infiltrate Haido Central Hospital to discover the whereabouts of Kir. He killed himself when Akai was hot on his trail.
- Ethan Hondou: A CIA agent who infiltrated the Black Organization. He committed suicide to protect his daughter Hidemi Hondou from being uncovered as a CIA agent.
- Scotch: An undercover operative who committed suicide before the Black Organization manages to reveal his true identity.
- Sherry (Ai Haibara/Shiho Miyano): A high ranking biochemist who worked on APTX 4869. She was scheduled to be executed when she boycotted her work because her sister Akemi was murdered. Shiho took an APTX 4869 pill she had smuggled in with her, with an intention to commit suicide, but instead shrunk to her 6-7 year old body. She then escaped. She has been living with Professor Agasa and working on an antidote to the APTX 4869, in order to restore Conan to his actual 'Shinichi body'.
- Rye: The codename of Shuichi Akai, he was an undercover FBI agent who infiltrated the Black Organization. His allegiance was discovered and he was rejected by the organization. His prowess led the organization to view him as the FBI's trump card.
- Ki'ichiro Numabuchi: A serial killer whom was slated to be the original test subject for the APTX drug before he escaped. He is currently in custody for murders he has committed, mostly in fear of the Black Organization sending agents after him. In the end, the court sentences him to death.
- An old man appeared during Shuichi Akai's final loyalty test while he was waiting for Gin to arrive. FBI agent Andre Camel emerged from his hiding place to scare him away, thinking he was just an ordinary old man. Since Gin never appeared, it was very likely the old man was a member of the Black Organization who was sent to scout ahead and reported that "Dai Moroboshi" was a spy.