Abstract: Natamycin is an antifungal antibiotic with high efficiency and broad spectrum, which demonstrates benign prospect of application in the field of medical care, food and health care as its efficient inhibition to the growth of fungus and fungal spores. In order to enhance the level of natamycin fermentation, Streptomyces natalensis was applied to produce natamycin by batch fermentation with shaking-flasks, research into single factors such as seed quality, components of fermentation medium and culture conditions was intensively conducted. Consequently, optimal fermentation medium that containing glucose 40 g/L, soya peptone 20 g/L and yeast extract 5 g/L with initial pH 7.0; culture conditions that the seeds cultivated for 48 h were inoculated into 250 mL flask containing 25 mL fermentation medium with an inoculum volume of 6% and fermented at 28℃ at 220 r/min were determined. Under these optimized conditions, the yield of natamycin was 1.472 g/L which was 150% higher than that before optimization, providing the foundation for relevant experiments.