Abstract: Methyl ivermectin and ethyl ivermectin, as a new kind of avermectin derivatives with high efficacy and low toxicity, were obtained from a genetically engineered strain Streptomyces avermitilis AVE-H39 and have a good prospect of development and application. Bioassay results showed that the insecticidal activity of ethyl ivermectin against the agricultural and forestry pests including Tetranychus cinnabarinus and Bursaphelenchus xylophilus was higher than that of methyl ivermectin. However, the industrial development of ethyl ivermectin is restricted by its low fermentation titer. In this research, a mutant strain with a higher fermentation yield of ethyl ivermectin was obtained through multiple rounds of mutagenesis and breeding. The fermentation medium was improved by Plackett-Burman experimental design and Box-Behnken design-response surface methodology (RSM). The optimized fermentation condition was corn starch 149.8 g/L, soybean flour 38.1 g/L, (NH₄)₂SO₄ 3.04 g/L, mannitol 30.0 g/L, yeast extract 20.0 g/L, CaCO₃ 3.0 g/L, CoCl₂ 0.01 mg/L, FeSO₄ 0.002 mg/L, rotation speed 220 r/min, initial pH 7.2, 30 mL medium per 250 mL Erlenmeyer flasks, inoculum volume 5%, fermentation temperature 28 ℃. Under the optimized condition, the fermentation titer of ethyl ivermectin reached the maximum value of 4965 mg/L. This research lay the foundation for the development of the new sixteen-membered macrolide insecticide ethyl ivermectin.