Hannigan's first major film role was in My Stepmother Is an Alien, a science fiction comedy released in 1988; one of her co-stars in the film was actor Seth Green, who would later join her in the regular cast of Buffy as her on-screen boyfriend. Then in 1989, her first regular role on a TV series came when she was cast in the short-lived ABC sitcom Free Spirit. Throughout most of the early-1990s, she appeared in commercials.

In 1997, Hannigan was cast to play Willow Rosenberg, Buffy's best friend, on the television series Buffy the Vampire Slayer (replacing Riff Regan, who played Willow in the unaired 25-minute pilot). The show became a success, and Hannigan gained recognition, subsequently appearing in several notable films aimed at teenage audiences, including American Pie, American Pie 2, Boys and Girls, and American Wedding. By the time Buffy had ended in 2003, Hannigan was earning a $250,000 salary for each episode. She also had a guest spot on the Buffy spin-off, Angel, reprising her role of Willow in a few episodes (including most notably "Orpheus," during the fourth season of Angel and the seventh season of Buffy), but none after Buffy finished production.

In early 2004, Hannigan made her West End debut, starring in a stage adaptation of When Harry Met Sally... at the Theatre Royal Haymarket, opposite Luke Perry. In 2005, Hannigan returned to starring in a regular television series, appearing in the hit comedy, How I Met Your Mother, as Lily, and also playing a recurring guest role on Veronica Mars as Trina Echolls. In February 2006, Hannigan starred as Julia Jones in Date Movie, a parody on romantic comedies.

• "How I Met Your Mother" .... Lily Aldrin (77 episodes, 2005-2009)
       - Three Days of Snow (2009) TV episode .... Lily Aldrin
       - Benefits (2009) TV episode .... Lily Aldrin
       - Little Minnesota (2008) TV episode .... Lily Aldrin
       - The Fight (2008) TV episode .... Lily Aldrin
       - The Naked Man (2008) TV episode .... Lily Aldrin
        (72 more)
• Date Movie (2006) .... Julia Jones
• Farce of the Penguins (2006) (V) (voice) .... Hottie Penguin
• "Veronica Mars" .... Trina Echolls (3 episodes, 2005)
       - My Mother, the Fiend (2005) TV episode .... Trina Echolls
       - Hot Dogs (2005) TV episode .... Trina Echolls
       - Ruskie Business (2005) TV episode .... Trina Echolls
• "King of the Hill" .... Stacey Gibson (1 episode, 2004)
       - Talking Shop (2004) TV episode (voice) .... Stacey Gibson
• "That '70s Show" .... Suzy Simpson (2 episodes, 2004)
       - Won't Get Fooled Again (2004) TV episode .... Suzy Simpson
       - Suzy Simpson (2004) TV episode .... Suzy Simpson
• Buffy the Vampire Slayer: The Animated Series (2004) (TV) (voice) .... Willow Rosenberg
• Americana (2004) (TV) .... Andrea
• American Wedding (2003) .... Michelle Flaherty
  ... aka American Pie: The Wedding (Australia) (Europe: English title) (Singapore: English title) (South Africa: English title) (UK)
  ... aka American Pie - Jetzt wird geheiratet (Germany)
• "Buffy the Vampire Slayer" .... Willow Rosenberg (144 episodes, 1997-2003)
  ... aka BtVS (USA: promotional abbreviation)
  ... aka Buffy (USA: short title)
  ... aka Buffy, the Vampire Slayer: The Series (USA: long title)
       - Chosen (2003) TV episode .... Willow Rosenberg
       - End of Days (2003) TV episode .... Willow Rosenberg
       - Touched (2003) TV episode .... Willow Rosenberg
       - Empty Places (2003) TV episode .... Willow Rosenberg
       - Dirty Girls (2003) TV episode .... Willow Rosenberg
        (139 more)
• "Angel" .... Willow Rosenberg (3 episodes, 2001-2003)
  ... aka Angel: The Series (USA: long title)
       - Orpheus (2003) TV episode .... Willow Rosenberg
       - There's No Place Like Plrtz Glrb (2001) TV episode .... Willow Rosenberg
       - Disharmony (2001) TV episode .... Willow Rosenberg
• Buffy the Vampire Slayer (2002) (VG) (voice) .... Willow Rosenberg
• Beyond the City Limits (2001) .... Lexi