Parrilla was born in Brooklyn. Her father, Sam Parrilla (1943–94), was a Puerto Rican-born baseball player who played professionally for 11 seasons (1963–73), including one season with the Major League Philadelphia Phillies in 1970 as an outfielder. Her mother is an American painter of Sicilian descent who works in banking. Parrilla has one older sister, Deena, and a nephew named Sammy. She is also the niece of character actress Candice Azzara. Parrilla's parents divorced when she was four years old. She spent her first ten years living with her mother, and then lived with her father. During the time she lived with her father, he was too protective to allow her to attend a performing arts school, which delayed her acting career. Parrilla lived with her father until his murder in 1994, when she was 16 and he was 50. Her father was shot once in the chest by a 15-year-old female assailant at point blank range and later died from the wound. After the death of her father, Parrilla moved in with her mother in Burbank, California. Parrilla visited Granada in 2007 to learn Spanish. After high school she moved to Los Angeles and attended Beverly Hills Playhouse to study acting. She also studied voice for ten years. Parrilla then began to be cast in small parts and later on, larger ones.
In her early career, Parrilla appeared in several movies, including Very Mean Men (2000), Spiders (2000), Replicant (2001) and Frozen Stars (2003). She made her television debut in 1999, on the UON sitcom Grown Ups. In 2000, she joined the cast of the ABC comedy series Spin City, playing Angie Ordonez for one season. She left the show in 2001. After that she joined Donnie Wahlberg and Neal McDonough in the short lived crime drama Boomtown, for which she received the Imagen Award for Best Supporting Actress, for her portrayal of Teresa, a paramedic. Initially a success, Boomtown began to struggle, and Parrilla's character became a police academy rookie, to tie her more closely to the rest of the show. "Boomtown" was cancelled just two episodes into its second season.
Parrilla guest-starred in a number of television dramas, including JAG, Six Feet Under, Covert Affairs, Medium, The Defenders and Chase. She had a recurring role in 2004 as Officer Janet Grafton in NYPD Blue. In 2005, Parrilla took a recurring guest role on the fourth season of the Fox series 24 as Sarah Gavin, a Counter Terrorist Unit agent. After just six episodes, Lana was made a regular cast member; but in the thirteenth episode, her character was written out after she tried to thwart another character's promotion from temporary to permanent CTU head Michelle Dessler (Reiko Aylesworth).
In 2006, Parrilla starred in the NBC summer series Windfall alongside Luke Perry, fellow former 24 cast member Sarah Wynter, and Parilla's former Boomtown castmate Jason Gedrick. In 2007, she guest starred as Greta during the third season of ABC's Lost in the episodes "Greatest Hits" and "Through the Looking Glass" In 2008, she had a leading role on the Lifetime movie The Double Life of Eleanor Kendall, in which she played Nellie, a divorcee whose identity has been stolen. Also in 2008, she starred in the CBS summer series Swingtown as Trina Decker, a woman who is part of a Swinging couple. In 2010, Parrilla had a female lead role in the Jerry Bruckheimer-produced Miami Medical on CBS, which had a short run towards the end of the 2009–10 television season before it was canceled in July 2010. Windfall, Swingtown and Miami Medical were all canceled after 13 episodes.
In February 2011, she was cast as Regina Mills/Queen Regina, in the ABC adventure fantasy drama pilot, Once Upon a Time created by Edward Kitsis and Adam Horowitz. The series debuted in October 2011. The pilot episode was watched by 12.93 million viewers and achieved an adult 18–49 rating/share of 4.0/10 during the first season, receiving generally favorable reviews from critics.
Parrilla's performance also received positive reviews from critics. In 2012 and 2013, she was regarded as a promising contender for an Emmy Award in the Outstanding Supporting Actress in a Drama Series category, though she did not receive a nomination. She won the TV Guide Award for Favorite Villain and the ALMA Award for Outstanding Actress in a Drama Series in 2012. Parrilla also received a nomination for Best Supporting Actress on Television from the 38th Saturn Awards.
Parrilla became engaged to boyfriend Fred Di Blasio on April 28, 2013, while in Israel. The two were married July 5, 2014, shortly before Parrilla began filming the fourth season of Once Upon a Time. Parrilla confirmed the news on her Twitter account on August 1, 2014.
Gallery of behind the scene stills released to promote the actor.