In order to acquire bacillus with beneficial effects on Largemouth bass, 94 strains of Bacillus were isolated from the intestines of Largemouth bass. Four strains of Bacillus subtilis including K-3, K-12, K-13, and K-22 were selected according to their hemolytic characteristics and enzyme production capacity, because the four stains were not hemolytic and produce high amounts of enzymes. Studies on the biological characteristics showed that the four strains were strong biofilm-forming strains and sensitive to a variety of common antibiotics, among which K-3 and K-13 could antagonize Nocardia. The survival rate of the four strains in artificial gastric juice (pH=4) and artificial intestinal fluid for 2 h reached more than 80%. After feeding the four stains to largemouth bass for 42 days, the body weight increase in K-13 group was significantly higher than that in the control group. The intestinal protease and lipase enzyme activities in K-12 and K-13 groups were significantly higher than that in the control group. In addition, compared with the control group, the expression level of pro-inflammatory factors in the head kidney, spleen, and intestine of K-13 group was down-regulated, and the expression level of inflammatory factors was increased, indicating that the K-13 strain had the potential to reduce the occurrence of intestinal inflammation in Largemouth bass. Collectively, the Bacillus subtilis K-13 isolated in this study has the characteristics of high safety, strong enzyme production ability, antagonism of Nocardia, and growth promotion to Largemouth bass, which can be used as a candidate strain for the development of probiotic preparations for Largemouth bass culture.