In order to improve milbemycin productivity and fermentation stability, the cultivation process and inoculation standard of milbemycin-producing strain were studied. The results showed that the optimal primary seed culture conditions were as follows: inoculation volume 3% (spores culture time 8 d, spore suspension concentration 1×108-7×108 spores/mL), grown 41 h under 28 ℃. For scale-up, the primary seed after 30 h cultivation was inoculated into the medium of secondary seed and cultivated for another 43 h. The characteristics of the secondary seed culture were as follows: pH value 7.2, mycelium concentration 29%-33%, total sugar content 20 g/L and amino nitrogen content 292 mg/L. The mycelium concentration and the productivity were increased by 4% and 5.6% compared with the primary seed cultivation, respectively.