Gelatin extracted from tilapia (Oreochromis niloticus) skin was hydrolyzed with serial digestions in the order of Bromelain and Alcalase. The antioxidative activity in vitro of tilapia skin gelatin hydrolysates (TGH) was researched. The results showed TGH had strong su peroxide/hydroxyl radical scavenging ability and the IC50 value was 3.13 m g·mL-1 and 3.58 mg·mL-1, respectively. The TGH was fractionated in to five major types of TGHⅠ(＞10 ku), TGHⅡ(10-5 ku), TGHⅢ(5-3 ku), TGH Ⅳ(3-1 ku), and TGHⅤ(below 1 ku) using an ultrafiltration (UF) membrane bio reactor system based on the molecular weight cutoffs. The fraction of TGHⅤ, exhibited the highest superoxide scavenging activity, was further purified usin g consecutive chromatographic methods including gel filtration, ionexchange ch romatography, and reversephase high performance liquid chromatography. The pur ified peptide had a strong scavenging ability on superoxide and the IC50 v alue was 4.6 μg·mL-1. And the molecular weight of the pu rified peptide was between 311.3-932.8 u determined by MS.