Lee Sang-yoon was picked out of a crowd on the streets of Yeouido by his former agency director at the age of 24. Making his entertainment debut in 2005 in a Hite Beer commercial, he went on to star in several television series. Lee won Best New Actor at the 2010 MBC Drama Awards for Home Sweet Home.

In 2013, Lee graduated from the prestigious Seoul National University with a degree in Physics. He had entered as a freshman in 2000, but his acting career and mandatory military service had caused him to take several years of leave from SNU, resulting in his delayed graduation.

Lee starred in his first big screen leading role in the 2014 romance film Santa Barbara.