The invention relates to a culture medium for producing an antifungal substance by streptomyces fermentation and a preparation method thereof. The culture medium comprises the following components in concentration by mass: 0.3 to 1.0 percent of corn flour, 1.5 to 2.0 percent of glucose, 2.5 to 3.5 percent of soy bean cake flour, 0.3 to 0.6 percent of potassium dihydrogen phosphate, 0.5 to 1.0 percent of magnesium sulfate and the balance of water. By adopting the fermentation culture medium, the yield of the streptomyces hygroscopicus BS-112 antifungal substance is greatly improved; and the industrially common raw materials such as the corn flour, the soy bean cake flour and the like are used for production, so the production cost is reduced and the culture medium is suitable for large-scale production. The produced antifungal substance has broad-spectrum antifungal activity on main mildewing fungi such as aspergillus flavus in grains and feed and multiple domestic silkworm and plant pathogenic fungi, has low toxicity, and has high practical application value and broad market prospect on mildew-proof agents for the grains and the feed and biological prevention and control of domestic silkworm fungi diseases and plant fungi diseases.