Abstract: In order to obtain high-yield nattokinase(NK)strains,the enzyme activity was determined by fibrin plate method,Bacillus subtilis XZI125 was mutagenized by atmospheric pressure room temperature plasma mutagenesis system(ARTP). High-yield NK mutant strains were obtained by casein plate method and four antibiotic resistance screening methods:rifampicin,kanamycin,streptomycin and chloramphenicol.The results showed that three high-yield mutant strains A27,Ar41 and Ac35 were obtained by six rounds of shaking flask fermentation. The enzyme-producing activity of the three strains after 5 d of shaking flask fermentation was 23.0%,25.1% and 26.5% higher than that of the original strain(2490 IU/mL),respectively.Compared with casein plate screening method and 0.7 μg/mL rifampicin,50 μg/mL kanamycin,60 μg/mL streptomycin and 300 μg/mL chloramphenicol,antibiotic resistance screening method was more conducive to the screening of NK high-yield strains.