Objective To realize the high-value utilization of tuna by-products and prepare antioxidant peptides from them.Methods Antioxidant protein peptides were prepared from the dark meat of yellowfin tuna by stepwise enzymatic hydrolysis using protease. The degree of hydrolysis and DPPH radical scavenging rate were used as evaluation indexes. The preparation process was optimized through one-way and response surface experiments, and the antioxidant activities, amino acid composition, and relative molecular mass distributions of the peptides were analyzed.Results The optimal preparation process for the antioxidant peptides was enzymatic hydrolysis with flavor protease at 50 ℃, pH 7.00, enzyme concentration of 6 000 U/g, and digestion time of 4.00 h. In the two-step enzymatic digestion, pepsin was used at 55.69 ℃, pH 1.41, enzyme concentration of 3 200 U/g, and digestion time of 5.16 h. Under these conditions, the degree of hydrolysis of the protein peptide and the DPPH radical scavenging rate were the highest, reaching 21.55% and 85.89%, respectively. The semi-inhibition concentration (IC₅₀) for DPPH radical scavenging by the tuna dark meat flavor protease-pepsin protein peptide was 1.613 mg/mL. Additionally, the peptide contained eight essential amino acids, accounting for approximately 39.60%, and hydrophobic amino acids, accounting for about 35.13%. Its relative molecular mass was mainly concentrated below 3 000, comprising approximately 63.86%.Conclusion Protein peptides obtained from tuna by-products through stepwise enzymatic digestion possess excellent antioxidant activity and high nutritional value.