Tuna protein hydrolysate (TPH) was prepared by hydrolysis with Prolyve BS and fractionated by membranes process. The antioxidant activities of recovered peptide fractions were evaluated. Four novel antioxidant peptides that were isolated from nanofiltration retentate exhibited the highest antioxidant activity， using gel chromatography and reversed phase high-performance liquid chromatography. The amino acid sequences of isolated peptides were identified as Tyr-Glu-Asn-Gly-Gly (P2)， Glu-Gly-Tyr-Pro-Trp-Asn (P4)， Tyr-Ile-Val-Tyr-Pro-Gly (P7) and Trp-Gly-Asp-Ala-Gly-Gly-Tyr-Tyr (P8) with molecular weights of 538.46， 764.75， 710.78 and 887.85 Da， respectively. P2， P4， P7 and P8 exhibited good scavenging activities on hydroxyl radical (IC 0.41， 0.327， 0.17 and 0.042 mg/ml)， DPPH radical (IC 0.666， 0.326， 0.451 and 0.377 mg/ml) and superoxide radical (IC 0.536， 0.307， 0.357 and 0.115 mg/ml). P7 was effective against lipid peroxidation in the model system. The isolated peptides might be useful used as natural food additive in food industry and formulation of nutritional products.