The invention provides a bacillus subtilis culture medium for producing anti-microbial peptide and application thereof in producing the anti-microbial peptide. The fermentation medium is composed of, by weight, 0.5%-1.5% of soluble starch or corn starch, 0%-1.0% of polyvalent peptone or beef extract, 0%-0.5% of sodium chloride, 0%-0.3% of monopotassium phosphate, 0%-0.3% of dipotassium phosphate, 0.05%-0.15% of magnesium sulfate, 0.005-0.015% of calcium carbonate, 0.05%-0.15% of manganese sulfate and the balance being yellow serofluid, wherein the pH value is adjusted to 7.0-7.5. According to the method for producing the anti-microbial peptide, the cell number of bacillus subtilis in obtained fermentation broth is large, and the produced anti-microbial peptide is high in titer.