Objective To establish an effective PCR assay for leptospirosis detection, and applicate the assay in tree shrew, mongolian gerbil and gray hamster. Methods Sequence of leptospira was obtained from the NCBI Genbank, and primers were designed based on the sequences. The positive amplified fragments were sequenced to verify the reliability of the method. The samples from tree shrew, mongolian gerbils and hamsters were tested using this PCR method. Results The PCR method for detection of leptospirosis was successfully established. The positive rate of Leptospira was 8.33% in 60 samples of conventional tree shrews, 100% in 104 samples of the conventional Mongolian gerbils, and 0% in 60 samples of clean gray hamsters. Conclusions The establishment of this PCR assay is useful in the detection of leptospirosis in tree shrew, mongolian gerbil and gray hamster. The results of our investigation of leptospira infection levels of the three new experimental animals may promote their application in biomedical research.