[Objective] To improve avermectin yield, mutants of Streptomyces avermitilis 9-39 were screened and the fermentation medium was optimized. [Methods] Atmospheric and room temperature plasma (ARTP) technology was used to treat the spores of S. avermitilis 9-39. The method of streptomycin and kanamycin resistance combined with 96 deep-well plate screening was used for high-throughput screening of high-yielding mutants. Furthermore, based on single factor experiments, the fermentation medium for mutants was optimized by response surface methodology. [Results] A stable avermectin high-producing mutant K-1A6 was obtained and its avermectin production reached 4.22 g/L, 23.4% higher than that of the parent strain 9-39. Under the optimized medium fermentation, the yield of avermectin reached 5.36 g/L, 27.01% higher than that before optimization. [Conclusion] The yield of avermectin can be increased by ARTP mutagenesis, screening and fermentation medium optimization.