Abstract: To study the anti-fatigue effect of walnut proteolytic enzymes and to provide scientific basis for the development and utilization of its functional products. Walnut isolated protein (WPI) was extracted from supercritically extracted walnut meal, and the WPI was enzymatically digested by Dregea sinensis Hemsl. protease. The hydrolysis degree (DH) was used as an index to study the optimal conditions for the preparation of walnut peptides, and the effects of different molecular weights of walnut proteins on the alleviation of exercise fatigue in mice were also investigated. The mice were randomly divided into five groups, and the test samples were orally gavaged daily for one month. Finally, weight-bearing and exhaustion swimming experiments were performed to determine the serum urea nitrogen (BUN), lactic acid (LA), hepatic glycogen (HG), myo-glycogen (MG), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and superoxide dismutase (SOD) contents. SDS-PAGE gel electrophoresis showed that the WPI was significantly degraded by Dregea sinensis Hemsl. Protease. The one-way combined response surface experiment showed that the DH was up to 30.1% when the material-liquid ratio was 9:100, pH9.6, enzyme addition quality 9.4%, enzyme digestion time 180 min, and enzyme digestion temperature 63 ℃. HE staining showed that the enzyme extracted by the use of Dregea sinensis Hemsl. Protease did not have any toxicity side effects on mice. Compared with other groups, the enzymatic digests with molecular weight less than 3000 Da showed a decrease in LA and BUN content, an increase in HG and MG content, and a more significant prolongation of weight-bearing swimming time (P<0.05), and a significant increase in the activities of SOD enzyme and GSH-Px enzyme in serum of mice in each group in the sample (P<0.05). It indicated that walnut proteolytic digests could alleviate exercise fatigue in mice, and the effect of small molecular weight walnut proteolytic digests was more significant, which might be realized by regulating antioxidant activity in vivo.