Gwendoline Yeo was born in Singapore and is the niece of George Yeo, who served as Singapore's Minister for Foreign Affairs. She moved to California as a teenager and graduated with honors from St. Ignatius College Preparatory in 1994, then finished her BA from UCLA before turning 20—summa cum laude and Phi Beta Kappa. She also earned a diploma in Classical Piano from the San Francisco Conservatory of Music.

Her musical training, particularly her specialization in the guzheng (a traditional Chinese zither), led to a solo radio show on NPR-KCRW where she premiered her first two tracks, "Kiss My Grits" and "Lovers." Before acting took over, she was active in pageantry, winning Miss Asian America in 1995 and Miss Chinatown USA in 1998–1999, which opened doors to hosting gigs and commercial work.

On television, she's best known for playing Dr. Kelly Lee on General Hospital from January to March 2006, apparently the show's only OBGYN. She's also guest-starred in 24, JAG, NYPD Blue, The O.C., Hannah Montana, and Chuck. Fans often note her recurring role as Xiao-Mei on Desperate Housewives. Her film work includes Daughters of Joy, Night Skies, The Perfect Girl, and Broken Trail (where she played Sun Foy, called "number three" by Robert Duvall), plus The Jane Austen Book Club in 2007, where she played Dr. Samantha Yep.

On the voice acting side, she's voiced Paine in Final Fantasy X-2 and Kingdom Hearts II, Lady Deathstrike in X-Men: Next Dimension, Mercy Graves in The Batman, Li-en in Zatch Bell!, and various other characters. She's sometimes confused with voice actress Gwendolyn Lau.