Seo Hyun-jin debuted as the lead vocalist of K-pop girl group M.I.L.K in 2001. However, the girl group soon fell by the wayside due to fierce competition among manufactured bands, which led to one of the members quitting before the outfit completely disbanded in 2003.

After M.I.L.K. was dissolved, Seo attended Dongduk Women’s University, where she majored in applied music to keep her dream of a career in singing alive. As a solo artist she contributed songs to a few soundtracks and SM Town compilations. When she got a chance to perform in her first musical, The Sound of Music, in 2006, she saw it as a turning point to begin her acting career. Besides joining more theatrical productions, Seo shot a movie and a short drama with boy band DBSK (at the time she and the group were under the agency SM Entertainment).

Supporting roles followed in the TV police procedural H.I.T (2007) and queer film Ashamed (also known as Life Is Peachy, 2011), though Seo got noticed for her subtler performances in period dramas such as Hwang Jini (2006), particularly her career-making performance in The Duo (2011) as a neighborhood tomboy who later becomes a gisaeng. Seo played her first villain in Feast of the Gods (2012), as an overly ambitious chef caught in a rivalry. Seo was then cast in more leading roles in the period dramas The King’s Daughter, Soo Baek-hyang (2013), and The Three Musketeers (2014).

Seo has also frequently appeared in projects directed by her best friend, actress Ku Hye-sun, notably short film The Madonna and the feature film Magic, in which she had her first leading role.