Tamblyn first tv role was 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 starred in the pilot episode of the revived Twilight Zone series on UPN in 2002. Tamblyn became a household name playing Joan Girardi, a teenage girl who receives frequent visits from God, on the CBS drama series 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 was supposed to return to CBS in the 2007 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. Tamblyn's next project, The Unusuals, will premiere on ABC on April 8, 2009. She stars as NYPD homicide detective Casey Shraeger in this crime drama. Notable guest roles include Buffy the Vampire Slayer (playing Janice Penshaw, the best friend of Dawn Summers), as well as Boston Public, CSI: Miami, and Punk'd (where Ashton Kutcher and his crew members tricked her into losing someone else's dog).

Tamblyn launched her film career playing bit parts in her father's movies: Rebellious and Johnny Mysto: Boy Wizard. She also appeared in 1995's Live Nude Girls. Her first major film role was in 2005's The Sisterhood of the Traveling Pants, co-starring Alexis Bledel, America Ferrera, and Blake Lively. She also starred in its 2008 sequel The Sisterhood of the Traveling Pants 2. Her horror film career began with the opening scene of 2002's The Ring. 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.

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

• Beyond a Reasonable Doubt (2009) (post-production)
• The Unusuals (2009) (TV) (post-production) .... Det. Casey Shraeger
  
• Spring Breakdown (2009)
• The Sisterhood of the Traveling Pants 2 (2008) .... Tibby Tomko-Rollins
• The Russell Girl (2008) (TV) .... Sarah Russell
• Normal Adolescent Behavior (2007) .... Wendy
  ... aka A Story of a Teenager (Europe: English title)
  ... aka Havoc 2: Normal Adolescent Behavior (USA: DVD title)
• Spiral (2007) .... Amber
• Blackout (2007/II) .... Claudia
• Babylon Fields (2007) (TV) .... Janine Wunch
• The Grudge 2 (2006) .... Aubrey
• Stephanie Daley (2006) .... Stephanie Daley
  ... aka What She Knew (USA: TV title)
• The Sisterhood of the Traveling Pants (2005) .... Tibby
  ... aka The Sisterhood of the Travelling Pants (Australia)
• "Joan of Arcadia" .... Joan Girardi (45 episodes, 2003-2005)
       - Something Wicked This Way Comes (2005) TV episode .... Joan Girardi
       - Common Thread (2005) TV episode .... Joan Girardi
       - Spring Cleaning (2005) TV episode .... Joan Girardi
       - Trial and Error (2005) TV episode .... Joan Girardi
       - Secret Service (2005) TV episode .... Joan Girardi
        (40 more)
• "Without a Trace" .... Clare Metcalf (1 episode, 2003)
  ... aka W.A.T (USA: short title)
       - Clare de Lune (2003) TV episode .... Clare Metcalf
• "CSI: Miami" .... Senior Cadet Valerie Barreiro (1 episode, 2002)
       - Camp Fear (2002) TV episode .... Senior Cadet Valerie Barreiro
• The Ring (2002) .... Katie Embry
• "The Twilight Zone" .... Jenna Winslow (1 episode, 2002)
       - Evergreen (2002) TV episode .... Jenna Winslow
• Ten Minutes Older: The Trumpet (2002) .... Kate (segment "Twelve Miles to Trona")
• "Boston Public" .... Melissa Campbell (1 episode, 2002)
       - Chapter Thirty-Two (2002) TV episode .... Melissa Campbell