Abstract: In this study，grub antimicrobial peptides were prepared to solve the antibiotic resistance and drug residue problem caused by the abuse of traditional antibiotics and feed additives. Enzyme hydrolysis method was used to produce small molecular antimicrobial peptides which showed strong inhibitory activity against both Staphylococcus aureus（ S. A） and Escherichia coli（ E. coli）. The results showed that，trypsin was found to be suitable for the preparation of antimicrobial peptides. Orthogonal experiment was used to determine the optimum experimental conditions for the preparation of antimicrobial peptides using trypsin. Orthgonal experiment showed that when protease adding was 50.00 ku/g，hydrolysis duration was 90min，the ratio of substance to liquid was 1∶10，pH was 7.50，the diameter of antimicrobial circle reached the greatest value against S. A and E. coli which is 11.5 mm and 8.7 mm respectively. The stability tests showed that the optimum pH was 6.00，and it had a stable antibacterial effect in high temperature environment. The effect of salt concentration on antimicrobial activity was different for different experimental bacteria. The antibacterial peptide of grubs had good resistance against trypsin hydrolysis，but had no stability to pepsin hydrolysis. These results indicated that grub antimicrobial peptides had strong antimicrobial activity and could be used in the area of agriculture，medicine and food industry.