Abstract: Bacillus velezensis Vel-HNGD-F2 screened from pig intestinal microorganisms in our lab has a good antibacterial effect on Fusarium graminearum and is expected to be used for the prevention and treatment of crop diseases caused by F. graminearum. In order to increase the yield of antifungal components, the fermentation conditions of Vel-HNGD-F2 were optimized by response surface methodology. Firstly, five factors including fermentation temperature, initial pH value of fermentation broth, shaking speed, fermentation time and inoculation amount were selected to optimize the single factor conditions. According to the results of single factor experiment, three factors including fermentation temperature, initial pH value of fermentation broth and inoculation amount were selected to optimize the response surface. The results showed that the optimum fermentation conditions of Vel-HNGD-F2 were as follows:fermentation temperature 36.49℃, initial pH value of fermentation broth 7.14, inoculation amount 3.13%. The predicted antifungal rate of fermentation supernatant was 58.71%, and the actual antifungal rate was 58.49%. The antifungal substance of Vel-HNGD-F2 was crudely extracted by acid precipitation method, and the antifungal activity was detected. The stability of the crude extract was studied. The results showed that the crude extract had good antifungal effect on F. graminearum. The antifungal rate reached (63.21±0.94)%. In addition, the crude extract had good heat resistance and was not sensitive to ultraviolet irradiation. Therefore, it is speculated that its antifungal effect is related to the secreted lipopeptides. The bmyB, fenD, ituC, srfAA and bacA genes participated in biocontrol of Vel-HNGD-F2 were amplified by using five pairs of reported biocontrol gene-specific primers, and the amplification results were verified by sequencing. The results showed that the strain contained genes homologous to bmyB, fenD, ituC, srfAA and bacA, and it was speculated that Vel-HNGD-F2 may secrete these five lipopeptides. Meanwhile, the components of lipopeptide crude extract were analyzed by UPLC-Q-TOF-MS. The results showed that the antifungal substance was a mixture of surfactin, iturin and bacillomycin. The strain can produce a variety of lipopeptides, which has a good control effect on F. graminearum, and has application potential of biological control of fungal pathogens.