Yoo Ah-in made his acting debut in the 2003 KBS2 drama Sharp. In 2007, he appeared in the film Boys of Tomorrow, which won him two Best New Actor trophies. In 2008, he starred in the comedy thriller film Antique, in which he was named Best New Actor at the 11th Director’s Cut Awards. In 2012, he scored a Best Actor nomination at the 21st Buil Film Awards for his work in the coming-of-age film Punch. In 2014, he acted alongside Kim Hee-ae in JTBC’s Secret Love Affair, which gave him a Best Actor for TV nomination at the 50th Baeksang Arts Awards. In 2015, he was featured in the films Veteran and The Throne, which earned him numerous citations from different award-giving bodies. In 2016, he received the Best Actor honors at the 52nd Baeksang Arts Awards for his leading role in the SBS sageuk Six Flying Dragons. In 2018, he led the critically acclaimed psychological thriller film Burning along with Steven Yeun and Jeon Jong-seo.  He raked in Best Actor awards for his performance in the movie. In 2021, he picked up another set of Best Actor trophies for the indie crime drama film Voice of Silence.

Aside from being an actor, Yoo Ah-in also served as chief editor of the fashion magazine Tom Paper from 2014 to 2016. He also wrote for InStyle Korea and Movieweek. Additionally, he acts as the representative, curator, and creative director of Studio Concrete, which is a creative collective established in 2014.

Yoo Ah-in was diagnosed with a benign bone tumor in 2015. The diagnosis and his other injuries from filming resulted in his exemption from mandatory military service. He identifies himself as a feminist.