This study aimed to identify potential probiotic strains of Bacillus subtilis from healthy fish gut microbiota for application in aquaculture. The effects of dietary B. subtilis administration on growth performance, serum enzyme activity, immune gene expression, and disease resistance in darkbarbel catfish (Pelteobagrus fulvidraco) were investigated. The isolate, identified through gene sequencing and biochemical tests, demonstrated resilience to pH 3.0% and 6.0% bile, and exhibited extracellular protease, cellulose, lipase, and amylase production. Darkbarbel catfish were fed diets with varying B. subtilis concentrations (0 CFU/kg [T0], 10