Objective To optimize the enzymatic hydrolysis process for collagen peptides from Trionycis Carapax and investigate their in vitro antioxidant activity and cytoprotective effects against oxidative damage.Methods With collagen peptides from Trionycis Carapax as the raw material, enzymatic hydrolysis parameters were optimized by using Box-Behnken response surface methodology. Ultrafiltration fractionation was applied to obtain collagen peptide fractions of different molecular weights, and the in vitro free radical-scavenging capacity was assessed. The cytoprotective effects were evaluated using an H₂O₂-induced oxidative damage model in B16F10 cells.Results The optimal enzymatic hydrolysis conditions were as follows: acid protease as the enzyme additive, temperature of 44.899 ℃, solid-to-liquid ratio of 1∶20.3 (g/mL), enzyme amount of 4 970.527 U/g, pH of 4.038, and hydrolysis time of 15.419 h. Under these conditions, the collagen peptides exhibited 2,2-Diphenyl-1-picrylhydrazyl (DPPH)·and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS⁺) scavenging rates of 90.9% and 94.8%, respectively. Collagen peptides with a relative molecular weight of less than five had the strongest antioxidant capacity, with scavenging rates exceeding 60% for DPPH, ABTS⁺, and hydroxyl radical (OH). Additionally, they significantly increased cell viability (P<0.05), upregulated catalase (CAT) and glutathione peroxidase (GSH-Px) levels (P<0.05), and reduced lactate dehydrogenase (LDH) release (P<0.05).Conclusion Collagen peptides from Trionycis Carapax with a low relative molecular weight exhibit significant antioxidant activity and cytoprotective effects.