To increase the fermentation titer unit of neutral protease from Bacillus strains,we studied the synergistic effect of multiple kinds of Bacillus strains for preparing neutral protease by mixture-culture fermentation,and optimized the combination manner of strains and the composition of the fermentation medium by single-factor experiments,fractional factorial design,steepest ascent design and central composite design to construct a proper mixture-culture fermentation system,which contained Bacillus strain A...