O'Loughlin was born on 24 August 1976, in Canberra, Australia, of Irish and Scottish descent.His father is a physics and astronomy teacher in Sydney and his mother is a nurse.
O'Loughlin suffered from obsessive-compulsive disorder when he was a child. He enrolled at the National Institute of Dramatic Art (NIDA) in Sydney in 1999 and graduated in June 2002 after completing a three-year, full-time Bachelor of Dramatic Art program.
O'Loughlin began working in short films and fringe theatre as a teenager in Sydney. One of his first acting jobs was an extra in a commercial, playing a Marine. After graduating from NIDA (National Institute of Dramatic Arts), he began his career in Australian television and film productions. Some of his TV credits include roles in BlackJack: Sweet Science, Love Bytes and White Collar Blue.