Abstract: Objective: To investigate the optimal enzymatic hydrolysis process for peptides derived from the swim bladder of Basa catfish and to evaluate their protective effects against hydrogen peroxide-induced cellular senescence. Methods: Basa catfish swim bladder peptides (BBP) were prepared through enzymatic hydrolysis. Using the degree of hydrolysis and total reducing power as evaluation indicators, the optimal enzymatic hydrolysis conditions were determined via the single-factor experiments combined with response surface methodology. BBP-I, BBP-II, and BBP-III were isolated and purified using Sephadex G-10 gel column chromatography. Their physicochemical properties were characterized in terms of molecular weight distribution, amino acid composition, and in vitro antioxidant activity. A hydrogen peroxide (H₂O₂)-induced cellular senescence model was established using mouse fibroblast (L929) cells to evaluate the effects of BBP-I, BBP-II, and BBP-III on cell viability, β-galactosidase activity, reactive oxygen species (ROS) levels, antioxidant enzyme activity, and collagen secretion, thereby assessing their protective effects against H₂O₂-induced skin senescence. Results: Results demonstrated that when flavor protease was used as the experimental enzyme, the optimal enzymatic conditions were as follows: a substrate concentration of 4%, a pH value of 5.0, a temperature of 50.5 ℃, an enzymatic time of 3.9 hours, and an enzyme dosage of 5000 U/g. Under these conditions, the total reducing power of BBP was determined to be 0.426±0.011. The molecular weights of BBP-I, BBP-II, and BBP-III were 925, 809, and 640 Da, respectively. BBP-I exhibited the highest content of aromatic amino acids, whereas BBP-II contained a higher proportion of hydrophobic amino acids compared with BBP-I and BBP-III. All three active peptide fractions demonstrated effective scavenging activity against DPPH·, O₂⁻·, and ·OH radicals. Among them, BBP-I exhibited the strongest scavenging capacity at the same concentration. In addition, BBP-I, BBP-II, and BBP-III exhibited no cytotoxic effects. They effectively restored the viability of senescent L929 cells and significantly inhibited cellular senescence (P<0.05). These peptides also significantly reduced the levels of reactive oxygen species and malondialdehyde (P<0.05), while enhancing the activities of superoxide dismutase and glutathione peroxidase (P<0.05), thereby inhibited the occurrence of the oxidative stress response. Moreover, the three active peptides were found to upregulate the expression of type I and type III collagen (P<0.05), indicating their potential in delaying skin aging. Conclusion: BBP-I, BBP-II, and BBP-III derived from the swim bladder of Basa catfish, exhibited significant antioxidant and anti-cellular senescence activities, thereby providing a theoretical basis and technical support for the high-value utilization of aquatic by-products.