Abstract The aim of this study was to improve the growth performance, immune response and disease resistance of grouper, Epinephelus coioides by using probiotic, Bacillus subtilis E20. The percent weight gain (PWG) and feeding efficiency (FE) of grouper administered the probiotic B. subtilis E20 were calculated. Survival of B. subtilis E20 in the posterior intestines was determined using a specific primer pair of BPHYF/BPHYR, as were the non-specific immune parameters of grouper, and its susceptibility to Streptococcus sp. and an iridovirus when fish were fed diets containing B. subtilis at 0 (control), 104, 106, and 108 colony-forming units (cfu) g−1 up to 28 days. Results showed that grouper fed a diet containing B. subtilis at the levels of 104, 106, and 108 cfu g−1 had significantly increased PGW (203.0%, 229.6%, and 238.0%) and FE (1.15, 1.20, and 1.22) compared to control (191.8% and 1.0), and these directly increased in a dose-dependent manner with B. subtilis concentrations. B. subtilis was able to survive in the fish's posterior intestines during the feeding period. The survival rate increased in grouper challenged with Streptococcus sp. or an iridovirus when the fish were fed B. subtilis at 104, 106, and 108 cfu g−1 for 14 and 28 days, and it was higher at 28 days than at 14 days. After 28 days of feeding, the relative survival percentages of fish challenged with Streptococcus sp. and an iridovirus were 22.8, 40.9 and 45.5, and 21.7, 30.4, and 52.2, respectively. The phagocytic activity, respiratory bursts, and superoxide dismutase (SOD) level of head kidney leucocytes as well as serum lysozyme activity and serum alternative complement activity (ACH50) of fish fed diets containing B. subtilis at 104, 106 and 108 cfu g−1 were significantly and dose-dependently higher than those of fish fed the control diet for 28 days. We therefore recommend dietary B. subtilis E20 administration of 104 – 108 cfu g−1 to E. coioides to promote growth, and enhance immunity and resistance against Streptococcus sp. and an iridovirus. The best results were seen in the 108 cfu g−1 group fed for 28 days.