The rainbow trout (Oncorhynchus mykiss) is an important commercial fish. Studies have shown that probiotics can promote the healthy growth of fish. In this study, we assessed the impact of an endogenous Bacillus subtilis strain (RT-BS07) on the growth, survival and immunological response of juvenile rainbow trout. Additionally, the morphology of rainbow trout’s intestinal tissue was measured. Control (CT) and experimental groups (B) were fed with conventional and probiotic-supplemented diets (1.0 × 108 CFU per gram) for 42 days, respectively. Following feeding, a challenge experiment was carried out with Aeromonas hydrophila at 1.0 × 107 CFU/mL to determine the experimental fish’s tolerance to the bacteria. The results showed that the weight gain rate (WGR) in group B was 118.2%, which was significantly higher than that in group CT (54.86%) after 42 days. The height of intestinal villi and the number of goblet cells increased in group B, thus increasing the resistance of rainbow trout to pathogenic bacteria. The alkaline phosphatase (AKP) activity (U/mg prot) in the liver, spleen and intestinal tissues of group B was significantly higher than that in the CT group. The expression of interleukin-1β (IL-1β) in the intestinal tissue of group B was 6-fold higher than that in group CT. Furthermore, the expression of growth hormone-2 (GH-2) in the spleen was 3-fold higher than that in group CT. After 12 days of A. hydrophila injection, the survival rate was 40% and 70% in group CT and group B, respectively. Collectively, the present study demonstrated that the addition of endogenous B. subtilis strain (1.0 × 108 CFU per gram) to the feed can effectively promote the growth of rainbow trout and enhance immunity, which is beneficial to rainbow trout culture.