To explore the effect of addition of exogenous amino acids on Brevibacillus laterosporus S62-9 (BL) secreted Brevilaterin, the antibacterial activity of BL fermentation broth and the composition change of Brevilaterin were analyzed by agar diffusion method and high performance liquid chromatography, respectively. The results showed that the addition of L-valine, L-methionine and 2-oxo-3-methyl-butyric acid could increase the antibacterial activity of the fermentation broth, and the maximum antibacterial diameter could increase by 18.08%. Furthermore, the addition of exogenous amino acids could also change the components of Brevilaterin. The addition of L-methionine could induce BL only to secrete Brevilaterin B and C, and the relative ratio between the two was reversed. The addition of L-valine or 2-oxygen-3-methyl-butyric acid also promoted BL to secrete a variety of new components. In conclusion, adding exogenous amino acids could effectively improve the antibacterial activity of the fermentation broth and even change the components of antimicrobial peptides, which might provide a theoretical foundation for the artificial regulation of microbial synthesis of novel and efficient antimicrobial peptides.