The original strain ASAGF 2-G4 was applied to undergo Atmospheric Room Temperature Plasma (ARTP) mutagenesis of various doses and 2 mg/mL NTG compound mutagenesis of different action durations. Five high-yield mutant strains were obtained after preliminary screening through 96-orifice-plate fermentation and re-screening in shake flasks. At last, a high-yielding mutant strain 2-A9 was selected, which increased the yield by 30.92% compared with the original strain after genetic stability verification. A high-yield mutant ASAGF 2-A9 was applied as the starting strain, the experiment design Plackett-Burman was used to screen the factors affecting the fermentation media, with a conclusion that factor glucose, dextrin, cottonseed protein and corn starch is a significant factor affecting butenyl-spinosyns. The experimental design Box-Behnken was applied to optimize the factors significantly affecting the experiment Plackett-Burman. The optimal growing media components were deduced as follows (g/L): glucose 60.0, dextrin 25.0, cottonseed protein 25.0, coen steep dry powder 12.5, peptone milk 10.0, MgSO4 1.0, NaCl 2.0, CaCO3 5.0. It’s verified that the average output of butenyl- spinosyns is close to the predicted value, indicating that the model is feasible and effective.