Probiotics are gaining popularity both empirically and scientifically as eco-friendly alternatives in aquaculture. The present research proposed to examine the influences of selective probiotics on the growth, hematology, microbes, and morphology of the intestine of stinging catfish (Heteropneustes fossilis) in a biofloc system. Additionally, this study evaluated the benefit–cost ratio (BCR) of specific probiotics used in the biofloc system. Stinging catfish fingerlings (average weight 0.86 ± 0.26 g) were equally distributed (250 fingerlings/400 L water-filled PVC tank) into three treatment groups and reared for 16 weeks. Two commercial probiotics (CP-1 and CP-2) and one laboratory-developed probiotic were used in the study. The results showed significantly higher growth (weight gain and specific growth rate), feed efficiency, improved intestinal microbiota, and enhanced morphology in fish reared with laboratory-developed probiotics in the biofloc system. Moreover, indicators of increased immune responses, such as enhancements in the intestinal mucosal fold, width of enterocytes and lamina propria, and abundance of goblet cells, were also observed in fish reared with laboratory-developed probiotics. The BCR, which determined business profitability, was also highest for the laboratory-developed probiotics in the biofloc system. Therefore, the results suggest that laboratory-developed probiotics are economically viable and environmentally friendly growth stimulators for stinging catfish culture in a biofloc system.