The biological effects of physical and chemical mutagenesis inducements on neutral protease produced by Bacillus subtilis SHB 2010-1 were investigated. Six selective media were studied on screening the efficiency of high-yield strain. Skim milk agar was proved to be the excellent cell culture medium, with which the hydrolysis zone was easily observed. On the base of the corresponding death curves, death rate at 90-95% was selected to take two-round ultraviolet-diethyl sulfate mutation. The enzyme activities of mutant strains from each step were 1.49%, 10.99%, 38.77% and 59.68%，respectively, higher than the original strain. The results showed that complex mutagenesis performed a more significant effect than single factor. The mutant strain named DES-59 were finally screened with the maximum enzyme activity of 7307 U/mL in 50 mL fermentation medium after 60 h, and the cell density of 2.205×1010 CFU/mL. The nprE gene of the mutant DES-59 was the same as sequence in database, indicating that the improvement was not contributed by the structural modification of neutral protease A, but by some unstated sites.