Abstract: In order to explore the research and development of oyster in the field of medicine and food homology, response surface methodology was used to optimize the preparation process of oyster protease-depeptidase, and its effects on testosterone secretion and oxidative stress in mouse leydig cells were studied. Based on the investigation of hydrolysis degree as the evaluation index, a biomimetic enzymatic hydrolysis method was employed to optimize the preparation process of oyster protein enzymolysis peptides using response surface analysis, building upon the foundation of single-factor experiments. Simultaneously, a hydrogen peroxide (H₂O₂)-induced oxidative damage model was established using mouse testicular interstitial cells (TM3), and the effects of oyster protein enzymolysis peptides on testosterone (T) secretion and oxidative stress were investigated through assessments of cell viability, DAPI staining, testosterone secretion level, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content in TM3 cells. The results showed that the optimal enzymatic hydrolysis conditions for oyster protein enzymolysis peptides were as follows: Substrate-to-solvent ratio of 1:10 g/mL, gastric protease concentration of 1.1%, hydrolysis time of 1.0 h, pancreatic protease concentration of 2.1%, and hydrolysis time of 3.1 h. Under these conditions, the degree of hydrolysis was determined to be 39.43%±0.42%. Oyster protein enzymolysis peptides exhibited varying degrees of proliferative activity on H₂O₂-induced TM3 cells, significantly (P<0.05) increasing testosterone secretion, SOD enzyme activity, and reducing MDA levels in TM3 cells. The most pronounced effects were observed at a concentration of 200 μg/mL of oyster protein enzymolysis peptides. In conclusion, the optimization of enzymatic hydrolysis process using response surface methodology proved to be effective and feasible. Oyster protein enzymolysis peptides was found to extremely significant promote TM3 cell proliferation, increase testosterone secretion, enhance SOD enzyme activity, and reduce MDA levels (P<0.01).