The study assessed the impact of dietary Bacillus subtilis supplementation on the growth and immune response of catfish. A 2-month feeding trial included control groups (Gr1&Gr2) and Bacillus-treated groups (Gr3&Gr4) with 1X10 10 CFU/kg Bacillus subtilis. Bacillus-treated groups (Gr3 and Gr4) exhibited significant improvements in the last weight, gaining weight, SGR%, and condition factor to the Gr1 and Gr2 groups of controls (Gr1 and Gr2). Additionally, the Bacillus-treated groups exhibited considerably reduced levels of ammonia (NH3) and nitrite (NO2) at the end of the trial. Afterward, (Gr2&Gr4) were infected with Aeromonas hydrophila, and hematological, blood serum parameters, and Aeromonas hydrophila count were assessed. The highest erythrocyte, hemoglobin, and PCV values were observed in the Bacillus-treated group (Gr3). However, RBCs, Hb, and PCV decreased significantly after the pathogen challenge in Gr2 compared to the unchallenged group. A leucogram revealed slight changes in (Gr2, Gr3, and Gr4) compared to (Gr1). The phagocytic activity showed significant enhancement in (Gr3) and significant reduction in (Gr2) compared to other groups. The infected group (Gr2) had increased AST, ALT, urea, creatinine, and TNF-α, along with decreased catalase enzyme, total protein level, albumin, globulin, and lysozyme activity relative to the control group (Gr1). However, Gr4 exhibited significant improvements in all these parameters compared to Gr2. Bacterial load was higher in group (Gr2) but lower in group (Gr4). In summary, this study suggests that adding Bacillus subtilis to the diet may improve the health and growth characteristics of catfish.