Abstract:[Objective] [Methods] The fermentation parameters on the β-1,3-1,4-glucanase production by Bacillus subtilis D-6 in a batch fermentor (5 L) were optimized by statistical analysis using response surface methodology (RSM) based on Box-Behnken design (BBD) analysis of variance (ANOVA). [Results] The results showed that under the optimized conditions, with agitation, aeration, and pH at 500 r/min, 1.05 vvm and 5.08, respectively, the highest β-1,3-1,4-glucanase activity of 2 294.4 U/mL was obtained after 22 h of cultivation. [Con-clusion] This study proved that RSM could efficiently be applied for modeling of β-1,3-1,4-glucanase production in submerged fermentation.