Jo Seung-woo debuted as an actor with a starring role in the 2000 film Chunhyang, which earned him Best New Actor nominations at the 21st Blue Dragon Film Awards and the 37th Grand Bell Awards. In 2003, he drew praises for his sincere acting in the romance melodrama film The Classic. His career breakthrough came in 2005 when he played an autistic runner in the hit biographical film Marathon, which gave him numerous Best Actor trophies. In 2006, he led the film Tazza: The High Rollers, which scored him another set of Best Actor wins and nominations. In 2012, he made his television debut in the MBC sageuk The Horse Healer, which won him the Daesang as well as a Top Excellence Award at the MBC Drama Awards. In 2017, he acted alongside Bae Doo-na in the tvN series Forest of Secrets, which took home the Daesang for TV at the 54th Baeksang Arts Awards. He also received the Baeksang Best Actor trophy for his performance in the drama. The series returned for a second season in 2020, with him reprising his role. Aside from his work in TV and films, Jo Seung-woo also maintains a successful career in the musical theater scene.

Jo Seung-woo enlisted for his mandatory military service in December 2008 and was discharged in October 2010. He dated actress Kang Hye-jung from 2004 to 2007. The couple starred as lovers in the romance drama film Love Phobia in 2006.