Abstract: The preparation process of walnut dregs protein enzymatic bacteriostatic peptide with antibacterial activity was explored. In this experiment,walnut dregs,a by-product of walnut kernel after extraction of oil was used as raw materials to obtain walnut protein antibacterial peptide by enzymatic hydrolysis,the preparation process was optimized and the antibacterial peptide was further purified. The inhibitory effect of the antibacterial peptide against Escherichia coli,Staphylococcus aureus and Bacillus subtilis were tested,and the antibacterial peptide component with the best bacteriostatic effect was selected. The results showed that,the optimal conditions for the pepsin enzymatic hydrolysis process were:Temperature of 54 ℃,pH of 3.46,enzyme dosage of 5475.71 U/g,which exhibited the highest degree of protein hydrolysis,up to 13.99%. The separation and purification of enzymatic peptides were carried out by ultrafiltration technology,and four enzymatic peptides with different molecular weights. The bacteriostatic experiments showed that the antibacterial effect of H-Ⅱ(molecular weight 3~10 kDa)was significantly higher than that of other molecular weight ranges(P<0.05). This study would provide the data support for the in-depth research and the development of walnut meal.