Abstract: In this study, effect of culture medium formula on yield and antibacterial activity of the lipopeptides produced by Bacillus velezensis FJAT-45028 was investigated to obtain the optimal medium for production of the antibacterial lipopeptides. The crude lipopeptides were extracted by acid precipitation and alcoholysis. Their component and content were determined using a liquid chromatography coupled with a quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS). The antifungal activity of the lipopeptides was evaluated byusing Neofusicoccum parvum as an indicator, which is a rot-causing fungus of the Longan fruits. The results showed that the strain FJAT-45028 could yield lipopeptides in the twelve tested culture media. Moreover, content of the crude lipopeptides varied from 2.28 g·L^-1 to 4.86 g·L^-1, and the diameters of inhibition zone varied from 12.46 mm to 20.02 mm. The results of LC-QTOF-MS detection indicated that the lipopeptides produced by FJAT-45028 were composed by iturin, fengycin and surfactin, and their contents in the twelve culture media varied within the range of 0.46-63.49 mg·L^-1, 34.07-918.86 mg·L^-1, and 7.00-53.24 mg·L^-1, respectively. The correlation analysis indicated that the contents of iturin, fengycin and total lipopeptides were significantly positive correlation with the antifungal activities (P<0.01). Finally, an optimal medium formula for production of the lipopeptides with antifungal activities by B. velezensis FJAT-45028 was as follow: Sucrose 20 g·L^-1, yeast extract 20 g·L^-1, beef extract 15 g·L^-1, MgSO₄·7H₂O 0.6 g·L^-1, FeSO₄·7H₂O 9.0 mg·L^-1, according to the antifungal activities and yield of the lipopeptides. In conclusion,the suitable medium for production of the antifungal lipopeptides was established, which laying the foundation for the industrial production of the antifungal lipopeptides from the strain FJAT-45028.