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Tamblyn was born in Santa Monica, California, the daughter of Russ Tamblyn, an actor, dancer, and singer, and Bonnie Murray, a singer, teacher, and artist. Her paternal grandfather, Eddie Tamblyn, was a vaudeville performer. At the age of ten, she played Pippi Longstocking at the Santa Monica Alternative School House; her father's agent, Sharon Debord, was attending as a family friend and ended up convincing her father to allow Tamblyn to go on auditions. She now lives in California with her daughter Andrea.

Tamblyn was cast as Emily Bowen (later known as Emily Quartermaine) on the soap opera General Hospital, a role that she played for six years (from 1995 to 2001). She also appeared in the films Rebellious and Johnny Mysto: Boy Wizard (both co-starring her father), as well as in Live Nude Girls (1995) and in the opening scene of the 2002 horror film The Ring. Tamblyn also made a number of guest appearances on several television shows, including Buffy the Vampire Slayer, in which she played Janice Penshaw, the best friend of Dawn Summers, as well as Boston Public, CSI:Miami, and Punk'd (after being tricked by Ashton Kutcher and his crew members for losing someone else's dog).

She also starred in the pilot episode of the revived Twilight Zone series on UPN in 2002.

Tamblyn became well known for playing Joan Girardi, a teenage girl who receives frequent visits from God on the CBS drama series Joan of Arcadia. That role earned her both a Golden Globe and Emmy nomination in 2004. Tamblyn was also nominated for a Saturn award for Best Actress In a Television Series in both 2004 and 2005. She won in 2004. She was also nominated for awards at The Teen Choice Awards, The Golden Satellite Awards and The Young Artist Award for Joan of Arcadia. Tamblyn's father made several appearances as God in the form of a dog walker on the show, which ran from 2003 to 2005.

Tamblyn's first major film role was in 2005's The Sisterhood of the Traveling Pants, alongside Alexis Bledel, America Ferrera, and Blake Lively. She also starred in the 2008 sequel The Sisterhood of the Traveling Pants 2.

In August 2006, Tamblyn won the Bronze Leopard at the Locarno International Film Festival for her performance in the title role of Stephanie Daley. The film, which also won an award at the 2006 Sundance Film Festival, features Tamblyn as a 16-year-old who kills her baby moments after giving birth in the bathroom of a ski resort. She was also nominated for Best Actress at the Independent Spirit Awards. The film also stars Tilda Swinton and Timothy Hutton.

Tamblyn's also appeared in the Japan-set The Grudge 2, a sequel to The Grudge. The film, which also stars Sarah Michelle Gellar, was released on October 13, 2006, and debuted in the #1 spot at the North American box office. Tamblyn, who learned about Japanese culture while filming the role, agreed to star in the film because she had wanted to play the lead in a horror film.

Tamblyn will next appear in Spring Breakdown, also featuring Amy Poehler and Rachel Dratch, and Blackout, a thriller by Italian writer Gianluca Morozzi, in which her character will be trapped in an elevator for nearly 24 hours. Tamblyn was supposed to return to CBS in the pilot Babylon Fields, an apocalyptic comedic drama about the undead trying to resume their former lives. However, the network excluded the show from its Fall 2007 programming lineup, since it would compete with the network's other undead-themed drama, Moonlight.

In January 2008, Amber appeared in the Hallmark movie, The Russell Girl about a woman suffering from disease and mental anguish.

Tamblyn, a political liberal, attended the 2004 Democratic National Convention, and has publicly voiced her displeasure towards George W. Bush. She supported Sen. Hillary Clinton in the 2008 Democratic presidential primaries. She has been a vegetarian since 2005 (stating that what keeps her from becoming a vegan is her taste for cheese.)

In February 2005, Tamblyn was threatened by a stalker. He pledged to "torture her for a million years" and "drill things into her kneecaps," among other acts of violence. Frightened, Tamblyn phoned the police. A restraining order was issued forbidding the man from coming within 100 yards of her.

Tamblyn is an agnostic. She is the goddaughter of actors Dean Stockwell and Dennis Hopper and musician Neil Young. Her uncle is Larry Tamblyn, who was keyboardist in the 60s rock band The Standells. Stockwell and her father, Russ Tamblyn, appeared together in the film The Boy with Green Hair and in the Quantum Leap episode 'Thou Shalt Not...'

She is currently dating actor David Cross.