Oh was born in Nepean, Ontario, to middle-class Korean immigrant parents Joon-Soo (John) and Young-Nam, who had come to Canada in the late 1960s. Her father is a businessman and her mother a biochemist.[2] Oh grew up living on Camwood Crescent in the Ottawa suburb of Nepean, Ontario, where she began dancing and acting at an early age.[4] For example, at the age of 10, she played "The Wizard of Woe" in a class play, "The Canada Goose". Soon after graduating from the National Theatre School in 1993, she starred in a London, Ontario stage production of David Mamet's Oleanna. Around the same time, she won roles in biographical TV films of two significant female Chinese-Canadians: as Vancouver author Evelyn Lau in The Diary of Evelyn Lau (Oh won the role over more than 1,000 others who auditioned); and as Adrienne Clarkson in a CBC bio-pic of Clarkson's life.

Oh became even more widely known in Canada for her lead performance in the Canadian film Double Happiness, for which she won the Genie Award for Best Actress. Her other Canadian films include Long Life, Happiness & Prosperity and Last Night, for which she again won a Best Actress Genie. Oh also won FIPA d'Or Best Actress at Cannes Film Festival for her role in The Diary of Evelyn Lau.

Oh is most familiar to American audiences from her roles in the popular films Under the Tuscan Sun and Sideways. She considers Sideways to be one of the two best movies she has made, along with Evelyn Lau. On American television, she is renowned for her current role in the hit Abcmedical series Grey's Anatomy, for which she has won both a 2006 Golden Globe Award for Best Supporting Actress in a Series and a 2006 Screen Actors Guild Award for Outstanding Performance by a Female Actor in a Drama Series. In July 2007, she received her third Emmy nomination for her work on the series.

In 2006, she costarred in the film The Night Listener as "Anna," alongside Robin Williams and Toni Collette. Although Oh remains active in feature films, the critically acclaimed Grey's Anatomy remains her primary current occupation.

Oh was host of the 28th Genie Awards on March 3, 2008.

Oh and Sideways filmmaker Alexander Payne, were in a relationship for five years, including two years of marriage; they married on January 1, 2003. Payne and Oh separated in early 2005 and divorced in late 2006. Oh is currently dating musician Andrew Featherston.

Oh's brother, Ray, is completing a Ph.D. in medical genetics at the University of Toronto and Ontario Cancer Institute. Her sister Grace is a crown attorney and mother of two children in Vancouver, British Columbia.