Jung Woo launched his acting career in 2001 with the film Running 7 Dogs. The audience began to take notice of him after he played a cheeky villain in director Lee Sung-han’s debut film Spare in 2008. They then reunited in the 2009 action-drama movie Wish, which Jung Woo had written himself. His portrayal of the film’s lead character won him Best New Actor at the 47th Grand Bell Awards. In 2013, he joined the cast of the KBS2 family drama You’re the Best, Lee Soon-shin, which earned him the Best New Actor trophy at the KBS Drama Awards. That same year, he starred in the hit tvN series Answer Me 1994, which gave him an Excellence Award at the 3rd APAN Star Awards. He was also named Best New Actor at the 50th Baeksang Arts Awards for his work in the series. In 2015, he appeared in the musical drama film C’est Si Bon, which scored him the Fantasia Award at the 19th Bucheon International Fantastic Film Festival. In 2017, he acted alongside Kang Ha-neul in the film New Trial. In 2022, he led the crime film Hot Blooded.

In January 2016, Jung Woo married actress Kim Yumi, who he co-starred with in the 2013 film Red Family. They welcomed their first child in December 2016.