Abstract: Medium and conditions for the fermentation of Streptomyces longissimus SL01 (CGMCC No.9543) were optimized, and the stability of the antifungal substance was investigated. The single factor experiment and orthogonal experiment were used to optimize the ingredients of medium and suitable fermentation conditions in shake flask. To evaluate the results of optimization, the methods of disease extension inhibition on the branches and apple fruits in vitro was used. The stability of the active substance in SL01 was measured under the condition of different temperatures, pH values and ultraviolet radiation. The optimum ingredients of fermentation broth were 2% millet powder, 1% yeast extract and 0.15% MgSO4·7H2O. The optimal conditions were 28, initial medium pH=7.0, inoculum volume 2%, liquid volume 50 mL/250 mL, and rotation speed 180 r/min. By using the optimized formula, the control effect of the SL01 fermentation filtrate against Valsa mali on branches and apple fruits had increased by 39.86% and 10.15%, respectively. The inhibitroy activity of fermentation broth was stable under 40, it was also relative stable when pH values ranged from 6 to 10, and it was almost not affected after exposed to UV irradiation for 12 h. This study proves that the inhibitory activity of SL01 fermentation broth was significantly improved by media optimization.