Abstract: In order to investigate best condition for liquid fermentation of strain SY286 and increase its antagonistic substance productivity against Plasmopara viticola, the single factor experiments and orthogonal experiments were adopted to optimize the liquid fermentation medium and fermentation condition. The result showed that the optimum medium was glucose 10 g/L, beef extract 7.5 g/L, NaCl 2.5 g/L, KNO3 5 g/L. The optimal fermentation conditions were culture temperature 27℃, fermentation time 72 h, initial pH 7.0, medium capacity 90 mL/250 mL, inoculation rate 6%, and rotate speed 150 r/min. The inhibitory activity of strain SY286 fermentation solution increased by 6.98% under the optimum fermentation medium and culture conditions, it was up to 99.19%.