The character was created by Paul Dini and Bruce Timm and was originally voiced by Arleen Sorkin in Batman: The Animated Series and its tie-ins. The character was portrayed by Hynden Walch on The Batman television series. In the Birds of Prey series, she was portrayed by actress Mia Sara, and by Sherilyn Fenn in the pilot episode.

IGN's 2009 list of the Top 100 Comic Book Villains of All Time ranked Harley Quinn as #45.

Harley Quinn first appeared in the Batman: The Animated Series episode "Joker's Favor", as what was originally supposed to be the animated equivalent of a walk-on role; a number of police officers were to be taken hostage by someone jumping out of a cake, and it was decided that to have the Joker do so himself would be too bizarre (although he ended up doing so anyway). Dini thus created a female sidekick for the Joker. Arleen Sorkin, a former star of the soap opera Days of our Lives, appeared in a dream sequence on that series in which she wore a jester costume; Dini used this scene as an inspiration for Quinn. Dini had been friends with Sorkin since college, incorporated aspects of her personality into the character.

The 1994 graphic novel Mad Love recounts the character's origin. Told in the style and continuity of Batman: The Animated Series and written and drawn by Dini and Timm, the comic book describes Harley as an Arkham Asylum psychologist who fell for the Joker and becomes his accomplice and on-off sidekick/lover. The story received wide praise[citation needed] and won the Eisner and Harvey Awards for Best Single Issue Comic of the Year. The New Batman Adventures series adapted Mad Love as the episode "Mad Love" in 1999, making it the second "animated style" comic book adapted for the series (the other was Holiday Knights).

She becomes fascinated with the Joker while interning at Arkham, and volunteers to analyze him. She fell hopelessly in love nearly instantly with the Joker during their sessions, and she helped him escape from the asylum more than once. When the Joker is returned to Arkham in a battered and broken state after a battle with Batman, the sight of the injured Joker drives Harley to don the costume and become Harley Quinn, the Joker's sidekick and love.

After Batman: The Animated Series and The New Batman Adventures, Harley makes several other animated appearances. She appears as one of the four main female characters of the web cartoon Gotham Girls. She also made guest appearances in other cartoons in the DC animated universe, appearing in the Justice League episode "Wild Cards" (alongside the Joker) and the Static Shock episode "Hard as Nails" (alongside Poison Ivy).

The animated movie Batman Beyond: Return of the Joker takes place in the future, long after the events in Batman: The Animated Series, but it does include a flashback scene with Harley's career apparently ending by her falling down a deep pit during a battle with Batgirl. At the end of the movie, two twin juvenile girls who model themselves on the Joker are released on bail to their respectable-looking grandmother who angrily berates them verbally and physically for their activities — to which they answer: "Oh, shut up, Nana Harley!"

She appeared in World's Finest: The Batman/Superman Movie as a rival and foil for Lex Luthor's assistant Mercy Graves; each takes an immediate dislike for the other, at one point fighting brutally with each other as Luthor and the Joker have a business meeting. In the film's climax, Harley nearly kills Mercy when she duct tapes her mouth shut and tapes her to the top of a gigantic killer android, although she is rescued and untied by Batman and Superman.

The character proved so popular that she was eventually added to the Batman comic book canon (although she had already appeared in the Elseworlds Batman: Thrillkiller 62 in 1997). The comic book version of Quinn, like the comic book version of the Joker, is more dangerously psychotic and less humorously quirky than the animated series version. Despite her noticeably more violent demeanor, Harley did tend to show mercy and compassion from time to time. She notably stopped Ivy from killing the vigilante Thorn, instead convincing her to leave the heroine hanging bound and gagged from a large statue.

Quinn's DC Universe comic book origin, revealed in Batman: Harley Quinn (October 1999), is largely an adaptation of her animated origin from the Mad Love graphic novel.