The effect of antibiotic-resistance screening on breeding of spinosad high-producing strains was researched by leading streptomycin, gentamicin, rifampicin and chloramphenicol into strain ASAGF W2 with two approaches.The first one was inducing different resistance to antibiotics through antibiotic plates successively, which was marked as non-GYM group. The second one was inducing next kind of resistance to antibiotic by antibiotic plates after the mutants was isolated and purified through streak plate method, which was marked as GYM group. All mutants were bred by the shake flask culture. The results show that non-GYM group is more suitable method for antibiotic resistance screening. Four spinosad high-producing strains have been obtained through five rounds screening and mutant 13-8-1 was obtained from isolates of antibiotic combination Str0.5Gen10 from non-GYM group and showed enhanced spinosad production by 23.87%, compared to original strain. The approach to screen spinosad high yield strains using multiple antibiotic-resistance screening is effective and easy in operation.