Abstract: In order to explore the best conditions for humic acid production from corn straw fermented by Bacillus subtilis, the proportion of corn straw and bran, the additive amount of urea and the initial pH of culture medium were taken as the experimental factors, and the content of humic acid was taken as the index. The factor levels were selected according to the results of single factor experiment, and the method of Box-Benhnken experimental design was used to study the interaction of various factors and their effects on humic acid, thus to obtain the quadratic polynomial regression model. The results showed that the optimal conditions of humic acid production from corn straw fermented by Bacillus subtilis were as follows: the proportion of corn straw and bran was 0.95∶1, the additive amount of urea was 0.81%, and the initial pH of culture medium was 6.1. After 7 days of fermentation at 37℃, the humic acid content of the fermentation products was 19.58%, which was 38.3% higher than that before the optimization.