Abstract: In order to investigate best condition for liquid fermentation of Bacillus amyloliquefaciens HF-01 and increase the antagonistic substance productivity of strain HF-01, the single factor experiments and orthogonal experiments were adopted to optimize the liquid fermentation medium and fermentation conditions. The optimum medium was 3.0% glucose, 0.5% yeast extract powder, 0.3% NaCl and 0.1% MgSO₄. The optimal fermentation conditions were initial pH 6.0—6.5, temperature 28 ℃, fermentation time 48 h, medium capacity 90 mL/250 mL, inoculation rate 6%, and rotate speed 130 r·min^-1. Inhibitory activity of strain HF-01 fermentation solution increased 37.3% under the optimum fermentation medium and culture conditions. When optimized filtrate was treated on citrus fruits under 25 ℃ for 4 days, the disease incidence and lesion diameter were 31.7% and 25.9 mm respectively, significantly lower than unoptimized filtrate with 56.7% disease incidence and 48.1 mm lesion diameter.