Abstract: In order to screen out pig gut-derived Bacillus subtilis with excellent biological activity, and to provide excellent bacterial strain resources for the development of differentiating functional microecological preparation, in this experiment, heat-treated healthy pig intestinal contents were used as the isolation matrix and Staphylococcus aureus as the indicator bacteria, and strain screening was carried out by bile salt tolerance isolation, indicator bacteria confrontation culture and enzyme production test. The morphological characteristics of the strain and the 16S rDNA gene sequence were identified. In addition, the agar diffusion method was used to investigate the bacteriostatic spectrum of the supernatant of strain fermentation, and tolerance and hemolysis on temperature, bile salt, pH value, artificial gastric fluid and intestinal fluid. The results showed that there were multiple strains in the intestinal contents that could inhibit indicator bacteria and enzyme-producing bacteria. Among them, 2 strains not only had good bacteriostatic performance, but also had the activities of protease, amylase, cellulase and xylanase, in which N65 strain had the best bacteriostatic performance and enzyme-producing activity. The N65 strain was closely related to Bacillus subtilis, and based on its morphological characteristics, it was identified as Bacillus subtilis. The N65 strain fermented supernatant had good inhibitory effect on Staphylococcus aureus, Clostridium perfringens, Streptococcus agalactiae, Escherichia coli, and Salmonella enterica subsp. enterica serovar Typhimurium, with a wide antibacterial spectrum, and had good tolerance on temperature, bile salt, pH value and artificial gastric fluid and intestinal fluid, and had low hemolysis rate, with no risk of hemolysis. The results suggested that pig gut-derived Bacillus subtilis N65 had good tolerance, bacteriostatic and enzyme-producing properties, and had the potential to be developed as a functional microecological preparation for feed antibiotic replacement.