The purpose of this experiment was to investigate the effect of low-fishmeal diet with Bacillus subtilis on growth performance, digestive enzyme activity, antioxidant enzyme activity and its mRNA expression of juveniles Epinephelus lanceolatu♂×E. fuscoguttatus♀. A total of 720 grouper with average initial weight of (7.00±0.02) g were randomly assigned into 8 groups in triplicates with 30 individuals per replicate. Eight diets were prepared by adding 0 (normal fishmeal control group), 0 (low fishmeal control group), 0.10%, 0.25%, 0.50%, 0.75%, 1.00% and 1.25% Bacillus subtilis in a diet with low fishmeal (15%), and were formulated by an optimum fishmeal group (35%) as positive control group. The results showed that there was no significant difference in survival rate (SR) among all the treatments. The weight gain rate (WGR) and specific growth rate (SGR) were increased firstly and then decreased with the B. subtilis level increasing. The feed conversation ratio (FCR) in 0.75% group has the lowest value, while the protein efficiency ratio (PER) showed the opposite trend compared to FCR. The activities of trypsin (TRY) and amylase (AMY) firstly increased and then decreased. The activities of serum catalase (CAT) and superoxide dismutase (SOD) in 0.75% group has the maximum value. The highest CAT and glutathione reductase (GR) mRNA expression in liver of fish were observed in 0.50% and 0.75% group, respectively. Grouper were challenged by Vibrio harveyi for the next 7 days, the survival rate of grouper increased significantly with the B. subtilis level increasing. It can be concluded that the supplementation of 0.63% B. subtilis (1.0×10⁸ cfu/mL) can significantly enhance the growth performance, disease resistance, digestive enzymes, serum CAT and SOD activity and mRNA expression related to immunity of grouper, which is estimated with WGR as the evaluation index.