Cho was born in Seoul, South Korea, and raised in Los Angeles, California, where his family settled in 1978. His father is a minister. Cho graduated from Herbert Hoover High School in Glendale, California in 1990. He then attended the University of California, Berkeley, graduating in 1996 with a BA degree in English and proceeding to teach English for some time at Pacific Hills School in West Hollywood, California. He is married to actress Kerri Higuchi. They are currently expecting their first child, a boy.

Cho's big break was a small role in the 1999 comedy American Pie, in which he popularised the slang term "MILF". His most successful starring role to date was as Harold Lee in 2004's Harold & Kumar Go to White Castle. Cho was one of the stars of the short-lived sitcom, Off Centre, and guest-starred on Charmed as a ghost. He was also a co-star of the now-defunct Fox sitcom Kitchen Confidential. Cho also had supporting roles in Bowfinger, American Beauty, Evolution, and Down to Earth.

In 2002, Cho had a starring role in the ensemble cast of Better Luck Tomorrow, a drama/dark comedy focusing on the travails of a group of Asian Americans living in Southern California who are academically successful, but socially discontented, and as a result engage in wantonly violent, criminal behavior. He was cast in NBC's new comedy The Singles Table September 2006, but the series never aired due to changes in scheduling and production. Cho was selected People Magazine's 11th sexiest man alive in 2006.

ho appeared on July 2004 issue of KoreAm Journal. He is the lead singer for Viva La Union,[4] a Los Angeles band composed of former UCB and USC students. In 2007, Cho was added to the cast of Ugly Betty as a recurring regular. Cho plays Kenny, a best friend of accountant Henry Grubstick (played by Christopher Gorham). Cho has been cast as helmsman Hikaru Sulu in J. J. Abrams's upcoming Star Trek movie.

Cho appeared in Nas "Be a Nigger Too" music video along with various celebrities.

In 2005, he appeared in NCIS as Marcos Siazon a contract assassin to kill a married couple who were Contract Killers, and Tony and Ziva were undercover as. When the real killers died in a car crash.