Lee Seo-jin made his small screen debut in the 1999 SBS series House Above the Waves. In 2001, he was named Best New Actor at the MBC Drama Awards for his work in Her House. He came to prominence in 2003 when he acted alongside Ha Ji-won in the MBC sageuk Damo, which won him an Excellence Award at the MBC Drama Awards. In 2004, he starred in the MBC romantic drama Phoenix, which gave him a Top Excellence Award at the MBC Drama Awards. In 2007, he took home another Top Excellence Award at the MBC Drama Awards for his portrayal of the titular character in the hit sageuk series Yi San. His performance in the drama also earned him a nomination for Best Actor at the 44th Baeksang Arts Awards. In 2016, he was paired with UEE in the MBC weekend series Marriage Contract, which scored him his third Top Excellence Award at the MBC Drama Awards. Aside from acting, Lee Seo-jin is also popular for his appearances in reality shows like tvN’s Grandpas Over Flowers and Three Meals a Day.

Lee Seo-jin graduated from New York University with a degree in business management. He previously dated his Lovers leading lady Kim Jung-eun.