Harley Quinn first appears in The Batman season 4 episode "Two of a Kind" voiced by Hynden Walch.
This version of the character is originally the host of a pop psychology television show called "Two of a Kind".
Claiming to have obtained an online degree for psychology, she gives off-the-cuff advice to her callers that usually hurts more than helps, which has created controversy, much to her boss Jimmy Herbert's discomfort. It wasn't until one Valentine's Day special, Harley sets up an ambush on Bruce Wayne to have him talk about a previous relationship with a woman named Kiki during her show, after falsely promising she would help him to promote a charity-drive for crime victims. Angered, Wayne leaves in a huff, and Herbert, finally fed up with Harley's irresponsible behavior, denounces Harley as a fake and pulls her show off the air permanently while apologizing to Wayne for the event.
The Joker, seeing the episode and learning that it led to an emotional wreck on Harley (as she is now left without a job), proceeds to take advantage of the situation. After meeting the despondent Harley in her apartment, he took her to his lair, where he admits of seeing her potential after watching her taming his two hyenas to sit. Initially thinking getting a tell-all from the Joker would restore her career, Harley Quinn ends up explaining her problems to him, and the Joker convinces to join him up in a rampage in Gotham under a costumed disguise to take out her rage. Convinced, Harley dons a jester costume and white makeup, being dubbed as 'Harley Quinn', and both wackos go around town creating chaos, something which the Batman finds it difficult to stop, even after he instantly learns of Harley's alter-ego.
Eventually, after escaping back to the Joker's lair after ignoring Batman's warning that the Joker is a psychopath, Harley learns that the Gotham Broadcast Network has gotten evidence of her alter-ego and are planning to send in a full profile of her on live. Angered, Harley plans to send the network down into pieces in revenge for her sacking, something which the Joker finds amusing and tags along with her. Breaking in to the studio, Harley plants up four bombs around it and holds the staff and audience hostage with the help from the Joker and his hyenas. However, Batman, along with Robin and Batgirl, arrive to the rescue and disable her detonation device. This causes Harley to accidently set off one of her bombs, rendering her unconscious.
As Robin and Batgirl get everyone outside the flaming building, the Joker and the hyenas escape, leaving Harley to die from the fire. Witnessing this, the Batman saves Harley from the fire before surrendering her to the police. It was then Harley seems to regret to taking up under the Joker's influence. However, once loaded into a police car, she sees that Joker has left her an affectionate message, and instantly falls in love with him again as she is hauled away to prison.
This incarnation of Harley Quinn wears a slightly different costume than the one she wears in Batman: The Animated Series, as the black parts of the original are dark red here, the headdress is larger and taller here than the original, the inverted diamond pattern on the shoulders and thighs is absent, and her mask conceals her eyes.
In this series Harley is on a more even footing as more of an ally/girlfriend as opposed to the henchgirl role from Batman: The Animated Series. She reappears in "The Metal Face of Comedy". Later she is captured by Batman, along with the Joker. "The End of Batman" is the last appearance of Harley Quinn as she and the Joker escape.