Abstract: In order to clarify the structure of antioxidant peptide from Pinctada fucata muscle by enzyme, the molecular weights of the fractions from reverse phase High-performance liquid chromatography (RP-HPLC) purification of the hydrolysate was determined by the technology of matrix-assisted laser desorption ionization time of flight mass spectrometry and the structure of the components with strong antioxidant capacity was analyzed and determined through tandem mass spectrometry.The results showed that the hydrolysate was purified by RP-HPLC separation, got five antioxidant activity strong peptides component A, B, C, D and E, its relative molecular weight were 1022.54, 1334.60, 1494.75, 1085.60, 1288.82 and 1304.83u, respectively.The peptide of component C had the strongest scavenging activity for 1, 1-Diphenyl-2-picrylhydrazyl free radical (DPPH·) and its amino acid sequence was Val-Ala-Thr-Gly-Leu-Arg-Lys-Glu-Gly-Val-Val-Gly-Gly-ProArg.The relationship between structure and antioxidant activity of the antioxidant peptide from Pinctada fucata muscle were initially revealed.