Objective To develop a multiplex polymerase chain reaction(mPCR) assay for detection of four pathogenic dermatophytes[Trichophyton mentagrophytes(Tm), Microsporum gypseum(Mg), Microsporum canis(Mc), and Arthroderma simii(As)]in laboratory animals, which could be used rapidly and simultaneously for direct detection of those four pathogens. Methods We designed 5 specific primers according to 18S-28S rRNA sequences of the four pathogenic dermatophytes reported in Genbank. The four mPCR assays were established through optimizing the concentration of primers, dNTP, TaqDNA polymerase and the annealing temperature. After verifying the specificity and sensibility, this method was used to detect 15 hair samples with artificial infection and 260 samples taken from laboratory animals. Results This mPCR technique can distinguish the four dermatophytes by producing 192 bp(Tm),460 bp(Mg),290 bp(Mc)and 602 bp(As)fragments. The sensibility for detection of the four dermatophytes was 5.9 pg/μL, 6.6 pg/μL, 9.5 pg/μL and 5.1 pg/μL, respectively. The results of 15 artificial infection samples were accurate, and the results of 260 hairs samples were negative for the four fungi. Conclusions Our results suggest that the mPCR assay developed in this study can efficiently detect the four dermatophytes, is a useful and rapid technique for rapid detection of the pathogenic dermatophytes in laboratory animals.