Abstract: As a by-product of oil extraction, walnut meal was often used as feed or discarded, resulting in a waste of a large amount of high-quality protein resources. In this paper, for the high-value utilization of walnut meal resources, the highest content of glutelin was used as raw material to prepare walnut glutelin Bacteriostatic peptide by solid-state fermentation with Bacillus subtilis and alkaline protease. The polypeptide content and the diameter of the inhibition zone of Escherichia coli and Staphylococcus aureus were selected as the evaluation indexes for single factor test. The polypeptide content and the diameter of the inhibition zone of Staphylococcus aureus were used as the response values to optimize the optimal bacterial enzyme synergistic solid-state fermentation process parameters. The thermal stability of the prepared glutelin Bacteriostatic peptide was investigated. The results showed that the single factor test showed that the glutelin Bacteriostatic peptide had a certain inhibitory effect on Escherichia coli and Staphylococcus aureus. The optimal fermentation process parameters under the response surface test were as follows: Inoculation amount 5%, solid-liquid ratio 1:1.5, fermentation temperature 43 ℃, fermentation time 3 d, and enzyme dosage 400 U/g. Under the optimal process conditions, the peptide content reached 26.35±1.29 mg/g, and the diameter of the inhibition zone against Staphylococcus aureus was 15.50±1.53 mm. Differential scanning calorimetry (DSC) analysis showed that glutelin antimicrobial peptides had higher thermal stability than glutelin. Studies have shown that the Bacteriostatic peptide prepared by solid-state fermentation of walnut glutelin with enzyme has good Bacteriostatic effect and relatively stable structure.