In order to rapidly select strains for highly yielding Penicillium citrinum nuclease P1 from a large number of mutants obtained under ultraviolet mutagenesis conditions, a TB-RNA plate screening method is adopted and the influences of various factors on pink enzymatic hydrolysis rings of the Penicillium citrinum nuclease P1 on a TB-RNA plate are researched to optimize the screening formula, and obtained optimal reaction conditions comprise an ultrafiltration water system, the toluidine blue concentration being 2 * 10<-4> mol/L and the RNA addition concentration being 0.1%. A reaction is carried out in a 28 DEG C constant temperature Incubator according to the optimal formula for 12 h, and it is found that the enzyme activity of the nuclease is positively correlated with and the diameter of the pink enzymatic hydrolysis rings in a culture dish, so the optimal TB-RNA plate method can be used for high-efficiency screening of the strains for highly-yielding the Penicillium citrinum nuclease P1. Ultraviolet mutagenesis are carried out to obtain 116 optimal strains, a diameter ratio of the enzymatic hydrolysis rings to a bacterium colony of above 1.71 is adopted as an index to screen multiple strains, late-stage seed mother shake flask fermentation and fermentation shake flask verification are carried out, and a result proves that six strains are better than original strains, and the ferment enzyme activity is maximally improved by 23%.