The former Tudors actor will play Captain Hook in the ABC fantasy comedy-drama.
Hook will be a recurring character in the upcoming second season.
Creators Adam Horowitz and Edward Kitsis had previously teased that Captain Hook may join the series at the show's Comic-Con panel.
The Irish actor returns to ABC after he previously played the lead role in the pilot episode of drama Identity in 2011.
He also played Emmet Fitzgerald in Irish soap Fair City and medical drama The Clinic.
O'Donoghue made his big-screen debut in last year's The Rite opposite Sir Anthony Hopkins.
Once Upon a Time will return to ABC on September 30.
