Accumulation of feed waste from a long-term aquaculture practice has resulted in the deterioration of the aquaculture environment. Floating net aquaculture engineering of tiger grouper (Epinephelus fuscoguttatus Forsskål, 1775) on Bacillus subtilis supplementation in the diet is one of the solutions to overcome the deterioration of the aquaculture environment caused by the accumulation of dieting waste. The purpose of the research was to study the effects of B. subtilis supplementation in the diet on protein digestibility, the efficiency of diet utilization, growth, and activities of digesting enzymes of E. fuscoguttatus. The sampled fish has an average weight of 4.24 g ± 0.023 g per fish. Diet used in the study contained 45 % protein with the supplementation of various amounts of B. subtilis varying in (0, 2.5, 5, 7.5, 10, and 12.5) % per kg of diet as A, B, C, D, E, and F treatment, respectively. The results show that B. subtilis supplementation in the diet significantly affected on protein digestibility (ADCP), the efficiency of diet utilization (EFU), feed conversion ratio (FCR), the protein efficiency ratio (PER), relative growth rate (RGR), survival rate (SR) and activities of digesting enzymes of E. fuscoguttatus. In conclusion, the optimum amounts of B. subtilis in the diet on ADCP, EFU, FCR, PER, RGR, and SR were at (7.34, 7.36, 7.18, 7.5, 7.48, and 7.5) % per kg of diet, respectively.