Abstract: To investigate the effect of surfactin on the inhibition activity of Bacillus amyloliquefaciens against Staphylococcus aureus,the key gene srfAC for surfactin synthesis was knocked out from B. amyloliquefaciens HZ-12. The results showed that the inhibitory activity of the mutant B. amyloliquefaciens HZ-12ΔsrfAC against S. aureus was significantly improved,and the relative inhibition rate was increased by 64%. Furthermore,the medium carbon and nitrogen sources of engineering strain B. amyloliquefaciens HZ-12ΔsrfAC were optimized. Under 30 g/L of corn starch and 40 g/L of soybean meal,the biomass of B. amyloliquefaciens HZ-12ΔsrfAC reached 3.3×10¹⁰ CFU/mL,2.23 times than that without optimization. This study confirmed that srfAC gene knockout could significantly improve the inhibitory activity of B. amyloliquefaciens against S. aureus for the first time,and the medium for efficient cell growth was obtained. The results had improtant theoretical significance and potential application value.