Jo Seung-woo grew up in a musical family: his Father Jo Kyung-soo is a singer, and his older sister Jo Seo-yeon acts in musical theatre. Jo himself also dreamed of becoming a musical actor from an early age, however in 1999 while a student at Dankook University he was persuaded to join auditions for Im Kwon-taek's film Chunhyang, and he ended up winning the part from among a field of 1,000 actors. Chunhyang would screen as the first Korean film in competition at Cannes, although domestically it failed to attract much of an audience.

Jo did go on to appear in musicals after his film debut, acting in local productions Subway Line 1 and The Last Empress. Soon he was drawn back into the film industry, however, with a key supporting role in Wanee & Junah (2001), a villainous turn in H (2002), plus a leading role in Who Are U? (2002). In 2003, Jo acted in Kwak Jae-yong's romance The Classic opposite Son Ye-jin, receiving good reviews for his sincere acting. His popularity continued to grow, and in 2004 he appeared in Im Kwon-taek's 99th film Low Life.

Jo's breakthrough would come in early 2005 with the smash hit Marathon, where he played an autistic young man who only finds release in running. The film sold over 5 million tickets, and Jo won several awards for his performance including Best Actor at the 2005 Grand Bell Awards and Best Actor in the foreign film category at China's Hundred Flowers Awards. Nonetheless, he continued to pursue his career in musicals, with critically acclaimed appearances in Hedwig and the Angry Inch and Jekyll and Hyde. His success at pursuing both film and musicals make him an unusual case among contemporary actors.

Jo starred in Love Phobia (2006) opposite then-girlfriend Kang Hye-jung; both were praised for their acting, but the melodrama wasn't a commercial success. He then headlined Tazza: The High Rollers with Kim Hye-soo, the 2006 film adaptation of Huh Young-man's same-titled manhwa, which went on to become one of the biggest Korean blockbuster hits of all time. He followed that with Go Go 70s, about a rock and roll band during the height of the Park Chung-hee military regime; and The Sword with No Name, in which he played a fictional royal guard in love with Empress Myeongseong (Soo Ae).

After completing his mandatory military Service, Jo made his comeback in the 2010 production of Jekyll and Hyde. The musical was especially meaningful to Jo since the actor rose to stardom when the show premiered in Korea in 2004. Jo's much-anticipated return to the stage was marked with controversy following reports that his salary would be the highest for any musical theatre actor in Korean history. While producers feared that demands for similarly high fees could follow (which could eventually put them out of business), others said that the fee was justified, based on the hope that Jo would help spark a renaissance in a once-vibrant but now-stagnant musical theatre industry. And true enough, when tickets went on sale, the demand was so high that the online reservation server broke down after 15 minutes, with all of the performances in which Jo was scheduled to appear already sold out.

His 2011 Sports movie Perfect Game revisited one of the most exciting matches in Korean baseball history, between Choi Dong-won of the Haitai Tigers and Sun Dong-yeol of the Lotte Giants in the summer of 1987, which ended in a tie after being extended 15 innings; the rivalry between the two was further heated up by regionalism at the time with Choi representing the Jeolla Province and Sun, the Gyeongsang Province. Jo starred as Choi opposite Yang Dong-geun as Sun. That same year, Jo also took on the lead role in the musical Zorro.

Jo made his small screen debut in 2012 with The King's Doctor, a period drama based on a true story about a Joseon-era low-class Veterinarian specializing in the treatment of horses who rises to become the royal physician. Jo won the highest award ("Daesang," or Grand Prize) at the MBC Drama Awards for his performance in The King's Doctor, then returned to the stage in 2013 in Hedwig, reprising one of his most memorable musical roles.

Jo reprised one of his most beloved roles in Jekyll and Hyde for the musical's 10th anniversary in late 2014, and the 18,700 tickets sold out in just 10 minutes. Because of his ticket power, he was chosen as among the top 30 most influential people in Korean popular culture in 2006, and for four consecutive years in 2010 to 2014.

Jo next plays a heroic prosecutor who uncovers bribery in the halls of power in Inside Men, a 2015 film adaptation of Yoon Tae-ho's webtoon The Insiders. Inside Men was a box office success with more than 7 million admissions, becoming Jo's highest-grossing film.

In his interview in July 2016, Jo said that after completion of his performance for the musical Sweeney Todd, he decided to take a three-year hiatus from musical to focus on film.

In 2017, Jo starred in tvN's mystery thriller drama Stranger, playing a prosecutor who lacks empathetic abilities. The series was a hit and gained favorable reviews for its tight plot, gripping sequences and strong performances.

In 2018, Jo is set to star in period film Grave Site, the third installment of the "divining art trilogy" by Han Jae-rim.