Objective: To improve the yield and antibacterial rate of antimicrobial peptides from Pleurotus tuber-regium mycelium (PTRM-AMPs). Methods: Taking the yield and the antibacterial rate of PTRM-AMPs against Staphylococcus aureus and Esche-richia coli as evaluation indexes, on the basis of protease screening, the extraction process of PTRM-AMPs was optimized, and the minimum inhibitory concentration of PTRM-AMPs was determined. Results: The optimum technological conditions for the preparation of PTRM-AMPs by alkaline protease hydrolysis were as follows: the enzymatic hydrolysis temperature was 39.5 ℃, the enzymatic hydrolysis time was 3.2 h, the pH value was 11.1, the solid-liquid ratio was 1∶20 (g/mL), and the enzyme addition was 6 000 U/g. Under these conditions, the actual antibacterial rates of PTRM-AMPs against S. aureus and E. coli were 74.17% and 86.22% respectively, and the yield of PTRM-AMPs was 22.67%. The MIC values of PTRM-AMPs against S. aureus and E. coli were 5.0, 2.5 mg/mL respectively. Conclusion: PTRM-AMPs has significant antibacterial activity, especially against E. coli.