She was primarily raised by her grandmother, Eva Flores Ruth, a vocalist who performed with salsa legend Tito Puente. DeLisle was heavily into goth bands like The Cure, but her mother, who had become a born-again Pentecostal, set a strict rule forbidding secular music. In her late teens, she started singing old gospel tunes, and entered the world of stand-up comedy on the advice of a close friend. In her comedy routine, DeLisle imitated voices very well, and was advised to take a shot at voice acting. Her first starring role came in the first English version of Crayon Shin-chan, as the voice of characters Georgie and Uma, in 1994.

Billed as a "Superstar Voice Actress" on the Anime News Network, DeLisle currently plays the leading role in at least six current animated series. Harp has called DeLisle "golden-voiced" and recognized her for her roles in The Fairly Oddparents as well as Clifford the Big Red Dog and its prequel, Clifford's Puppy Days. She is the most recent actress to voice Daphne Blake in What's New Scooby Doo?. In addition to her numerous English-speaking roles, she also speaks Japanese as the character Yumi on Hi Hi Puffy AmiYumi. DeLisle has had voice acting roles in many computer and video games, such as Escape from Monkey Island, Baldur's Gate II: Shadows of Amn, Lara Croft Tomb Raider: Anniversary, and several Star Wars games. As a voice actress, she portrays the Dark Jedi, Asajj Ventress, in 5 episodes of Star Wars: Clone Wars and reprises her role as Ventress in a bonus level in the Force Duel mode of the PSP version of the video game Star Wars: The Force Unleashed. As a voice actress, she portrays Azula on Avatar: The Last Airbender, several voice roles (though mainly Mandy) on The Grim Adventures of Billy & Mandy, Therese and Jeanette (Tourette) Voerman in the White Wolf RPG PC game Vampire: The Masquerade – Bloodlines, Frida Suárez in El Tigre: The Adventures of Manny Rivera, Sam Manson in Danny Phantom, as well as Vicky in The Fairly OddParents in Xiaolin Showdown as Kimiko Tohomiko and also in W.I.T.C.H as Miranda .

Her full range of vocal talents was on display in the cartoon Batman: The Brave And The Bold episode "Mayhem of the Music Meister!" The characters were driven to burst into song by the Music Meister; DeLisle provided the voice for the heroine Black Canary singing about her unrequited love for Batman, and later Green Arrow. She had a role in Transformers: Revenge of the Fallen as the arcee triplets.

DeLisle has released four music CDs, which include goth-inspired folk and Americana tunes, and a few gospel-style songs.

In 2002, DeLisle married musician Murry Hammond, the bassist for the Old 97's, after a few months in a long-distance relationship. They were featured in an episode of the TLC program A Wedding Story. Their first child, Jefferson Texas "Tex" Hammond, was born on January 31, 2007 in Los Angeles, California.

Grey Delisle is also known for her work in the Star Wars universe, As a voice actress, she portrays the Dark Jedi, Asajj Ventress, in 5 episodes of Star Wars: Clone Wars and reprises her role as Ventress in a bonus level in the Force Duel mode of the PSP version of the video game Star Wars: The Force Unleashed. She played the bounty hunter Shara, love interest of William Beckman's character X-2, the clone of Jedi Knight Falon Grey in the video game Star Wars Battlefront: Elite Squadron. Voiced "Noble Youth" in Star Wars Knights of The Old Republic- Upper City Cantina.