Bonito protein hydrolysates were prepared by enzymatic hydrolysis using bonito fish as the raw material. Enzymatic hydrolysis processing was optimized using antioxidant activity and protein recovery rate as indicators. The protein hydrolysates were then separated and purified by means of ion exchange chromatography and gel chromatography. Finally, matrix-assisted laser desorption/ionization-time of flight-mass spectra (MALDI-TOF-MS), in tandem with reversed-phase high-performance liquid chromatography (RP-HPLC) was applied for structural identification of fraction composition. The optimum For the hydrolysis reaction, the best hydrolytic enzyme and its concentration were papain; and 1.0%, respectively. The optimum pretreatment conditions were 100 ℃ for 5 minutes After 4-hour hydrolysis, three fractions, labeled A, B, and C, were obtained from the hydrolyzate through DEAE-52 cellulose column chromatography. Fraction A had the highest antioxidant activity, which was further separated by gel chromatography on Sephadex G-15 column to give two fractions. The fraction with the highest antioxidant activity was labeled A1 and the other was labeled A2. Using MALDI-TOF-MS, the molecular weights of A1 and A2 were found to be around 378.830 Da. Further amino acid analysis revealed that the short-chain peptide had a possible amino acid composition of Lys, Leu, and Pro.