Abstract: Armillaria mellea Vahl ex Fr was obtained by enzymatic hydrolysis with Armillaria mellea Vahl ex Fr powder as raw material.The preparation process of walnut peptide was optimized on foundation of single factor experiment, according to Boxbehnken central composition design principle, the method of response surface analysis was adopted with the degree of hydrolysis as the response value.The effect of concentration of substrate, enzyme dosage, time and temperature on the degree of hydrolysis were studied in this paper.Fatigue model was established by swimming test.Effects of Armillaria mellea Vahl ex Fr Peptides on swimming time, liver glycogen, muscle glycogen, serum urea nitrogen, lactic acid, LDH were determined.The rusults showed that the optimum preparation conditions were obtained as follows: concentration of substrate 7.4%, enzyme dosage 2%, time 4.6 h and temperature 51 ℃, the degree of hydrolysis was 35.61%, which was close to the theoretical value. Take the middle dose group as an example, compared to the blank group, the Armillaria mellea Vahl ex Fr Peptides increased content of liver glycogen and muscel glycogen 109.01% and 58.33%, reduced metabolic product of LA and BUN levels 36.99% and 30.33%, increased LDH activities 9%, their differences were significant ( p < 0.05) . The Armillaria mellea Vahl ex Fr Peptides has the function of anti-fatigue obviously.