Acetobacter pasteurianum C9-4 was used as bran fermentation strain, and the preparation conditions of acetic acid bacteria bran koji fermentation were optimized by response surface method. The result showed that the best preparation conditions were: content of acetic acid 1.03 mL/100 g, condent of ethanol 2.09 mL/100 g and content of inoculation 4.09 mL/100 g. Under the condition, the production reached 1.38×10⁸ CFU/g. The culture was expanded in vinegar factory with the optimized parameter, the total number of acetic acid bacteria could reach 10⁸ CFU/g in the process of expanding the culture. This experiment provides a reference for the preparation of acetic acid bacteria bran koji and its bioaugmentation in solid vinegar fermentation.