The potential of Bacillus subtilis E20-fermented soybean meal (FSBM) as a partial alternative component of fish meal (FM) in fed diets of orange-spotted grouper (Epinephelus coioides) was evaluated in this study. An FM-based diet and seven diets containing 10%, 20% and 30% and 10%, 20%, 30% and 40% of FM replaced by soybean meal (SBM) and FSBM, respectively, were fed to grouper for 84 days to evaluate possible substitution levels of FM by tracking growth performance, digestive enzyme activity and morphological changes in the liver and distal intestine. No significant differences in survival and muscle composition of grouper were found between controls and treatments. Growth performance and feed efficiency of fish fed diets with FM replaced by FSBM up to 30% were not significantly different from controls, whereas significantly decreased growth performance and feed efficiency occurred with diets containing >20% of SBM. Based on the feed efficiency, the maximum substituted levels of FM by SBM and FSBM in grouper diets were 18.36% and 29.32%, respectively. Based on this study, the research group concluded that FSBM has great potential to be used as a protein source in grouper diets in partial replacement of FM.