Abstract: In order to optimize the fermentation process of nemadectin produced by Streptomyces cyaneogriseus ssp. Noncyanogenus, the fermentation conditions was primarily studied using the method of shaking flask. The effects of some factors, such as time, temperature, dissolved oxygen and inoculation volume, were demonstrated through single factor tests. The results showed that spores of S. cyaneogriseus ssp. noncyanogenus incubated for 8 d were applicable to seed preparation, and then the strain’s working cell bank was established by glycerol tube cryopreservation. But the save time should be no more than 2 months. While the strain in the working cell bank was inoculated into seed medium and then cultured at 28℃ for 28 h, the seed of better quality was obtained. The optimum fermentation condition of shaking flask was that the strain seed culture was inoculated into 50 mL of fermentation medium in the flask of 250 mL with the ratio of 5% and fermented at 25℃ for 8 d with shaking speed of 250 r/min. It indicated that nemadectin yield was up to 164.90 μg/mL.