In the present study, we aimed to screen the potential probiotic Bacillus subtilis isolated from the gut of healthy fish using in vitro assays and to evaluate its effect on Dabry's sturgeon (Acipenser dabryanus) using in vivo feeding experiments. Among the isolates, B. subtilis BSth-5 and BSth-19 exhibited antimicrobial effect against four sturgeon-pathogenic bacteria, including Aeromonas hydrophila, A. veronii, A. media, and Streptococcus iniae. The cell number of B. subtilis BSth-5 and BSth-19 changed little after 2 h of exposure to pH 3.0 or fresh Dabry's sturgeon bile at 2.5% and 5.0%. Meanwhile, B. subtilis BSth-5 and BSth-19 produced extracellular protease, cellulose, and lipase. And it was proved that B. subtilis BSth-5 and BSth-19 were harmless after injection of Dabry's sturgeon. One group of Dabry's sturgeon was fed a control diet and two groups were fed experimental diets containing 2.0 × 108 CFU/g BSth-5 (T1 group) or BSth-19 (T2 group) for 8 weeks. No significant differences in final weight, weight gain rate, and special growth rate were observed in the T1 and T2 groups compared to the control group (P > 0.05), but a significant improvement in survival rate was detected after 4 and 8 weeks of feeding (P < 0.05). After 8 weeks, serum total antioxidant capacity, total superoxide dismutase activity, and IgM levels were significantly higher in the T1 and T2 groups compared to the control group (P < 0.05). Moreover, serum lysozyme activity was significantly higher in the T1 group relative to the control group during the whole experiment period (P < 0.05); however, the differences were not significant between the T2 and control groups (P > 0.05). Serum malondialdehyde levels in the T1 and T2 groups were significantly lower than those in the control group after 4 weeks (P < 0.05). Sturgeons in the T1 and T2 groups showed a higher survival rate after Aeromonas hydrophila infection. To summarize, dietary supplementation with BSth-5 and BSth-19 could enhance the survival rate, antioxidant activity, serum immunity, and disease resistance in A. dabryanus.