Effects of Bacillus cereus BC‐01, Lactobacillus acidophilus LAG01, Clostridium butyricum CBG01 and their combinations as supplementation on the growth performance, digestive enzyme activities and serum non‐specific immunity of hybrid grouper (Epinephelus lanceolatus ♂×E. fuscoguttatus ♀) were assessed. Seven different diets, that is one control diet (basal feed without any probiotics, CT) and six treatment diets containing single B. cereus (Bs), L. acidophilus (Ls) and C. butyricum (Cs) at 1.0 × 109 cfu/kg feed, and also their combinations in equal proportion at 1.0, 2.0 and 3.0 × 109 cfu/kg feed (BLC1, BLC2 and BLC3) were prepared respectively. After 60‐day feeding trial, the final weight, specific growth rate,food consumption, food conversion efficiency and apparent digestibility coefficient of fish in Ls and BLC3 were significantly higher compared with the control (p < .05). The activities of pepsin and trypsin in the intestine of fish for Ls and BLC3 were significantly higher relative to the control (p < .05). Relative to controls, significantly enhanced amylase and lipase activities in proximal intestine except for Cs and BLC1 and lipase activities in distal intestine except for Cs were observed (p < .05). Meanwhile, activities of superoxide dismutase in the serum of fish for all treatments, lysozyme and catalase in Ls and BLC3, and glutathione peroxidase except for Cs were significantly enhanced (p < .05). Based on the above, dietary supplementation of single L. acidophilus at 1.0 × 109 cfu/kg or combination of three strains at 3.0 × 109 cfu/kg for hybrid grouper is recommended.