Jung Il-woo made his acting debut in the 2006 thriller movie The World of Silence. He then gained attention with his appearance in the 2006 MBC daily sitcom Unstoppable High Kick, in which he won the New Star Award at the 1st Mnet 20’s Choice Awards and the Best Male Newcomer trophy at the 2007 MBC Entertainment Awards. He was also nominated for Best New Actor for the film My Love at the 44th Baeksang Arts Awards in 2007. In 2009, he landed his first leading role in the MBC sageuk drama Return of Iljimae, which won him the Daesang at the 2nd Korea Junior Star Awards. His breakout role came in the 2011 hit SBS drama 49 Days, in which he played the role of a witty Grim Reaper. It was followed by a leading role in the 2011 tvN romantic comedy Flower Boy Ramyun Shop. In 2012, he became part of the sageuk The Moon That Embraces the Sun, in which he was nominated for Most Popular Actor for TV at the 48th Baeksang Arts Awards. In 2014, he won a Top Excellence Award at the MBC Drama Awards for the sageuk Records of a Night Watchman.

Jung Il-woo’s father is former television news anchorman Jung Hae-hoon. In 2006, he was involved in a car accident with his friend and fellow actor Lee Min-ho. The accident caused him to have partial memory loss. In December 2016, he pushed through with his mandatory military service even after being diagnosed with a cerebral aneurysm. He was discharged on November 30, 2018.