Spinosad high-producing strains were breeded from strains ASAGF 13-8-1 mutated by multifunctional plasma mutagenesis system（MPMS）, combined with streptomycin, gentamicin, chloramphenicol and rifampicin resistance screening. A fast and high throughput screening method of 96-well plate and bioassay as well as the shake flask culture were employed to primary and secondary screen respectively. One mutant 14-2 have been obtained from isolates of MPMS mutagenesis combined with antibiotic of 0.9μg/mL streptomycin,14μg/mL gentamicin,400μg/mL rifampicin,2μg/mL chloramphenicol through five rounds rescreening, and showed spinosad production enhanced by 28.68%, compared to original strain. By comparing the impact of MPMS mutagenesis and MPMS mutagenesis combined with antibiotic combination of Str 0.9 Gen 14 Rif 400 Chl 2 on ASAGF 13-8-1, the results showed MPMS mutagenesis combined with antibiotics help breed of spinosad high-producing strains.