Based on the study of sugar supplement process, high efficient fermentation with Streptomyces gilvosporeus HBJ591 was carried out via the optimization of the fermentation medium. When the reducing sugar concentration in the fermenter was 2.5%, the synthetic course of natamycin was extended effectively. Response surface method was used to optimize the fermention medium of HBJ591, then fed-batch fermentation was carried out in 16 L fermenter, the natamycin fermentation level reached 15.7 g/L, and the highest yield was 4.8 times higher than the original process.