This study evaluated the impact of Bacillus subtilis supplementation on growth, antioxidant status, digestive enzyme activities, hematological parameters, blood biochemistry, and immune responses in Nile tilapia (Oreochromis niloticus) fingerlings. A total of 180 fingerlings (mean weight: 5 ± 0.5 g) were randomly assigned to four groups: a control group and three probiotic-supplemented groups with 10⁶ (S-1), 10⁸ (S-2), and 10¹⁰ (S-3) CFU g⁻¹ of B. subtilis, following a completely randomized design. Fish were fed twice daily for eight weeks. Results showed that the 10¹⁰ CFU g⁻¹ group exhibited significantly higher weight gain (43 g), specific growth rate (2.5%/day), and better feed conversion ratio (0.7) compared to the control (30 g, 2.2%/day, and 1.4, respectively). Hematological parameters (WBC, RBC, hemoglobin) and immune responses (phagocytic activity, lysozyme) were significantly improved. Blood biochemistry showed decreased ALT/AST levels and increased ALP activity. Digestive enzymes (protease, lipase, amylase) and antioxidant activity (GPx, CAT, SOD) were enhanced, with reduced MDA levels. No significant differences were observed in survival rates or stress responses. These findings suggest that B. subtilis supplementation at 10¹⁰ CFU g⁻¹ improves growth, immune function, and antioxidant status, making it a promising probiotic for Nile tilapia.