Objective To compare the efficiency of bacteria culture and PCR assays for detection of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) in laboratory rats and mice. Methods Bacteria culture combined with biochemical identification and PCR assay were used to detect 78 SPF rats and 422 SPF mice and the results of the two methods were compared. Results All the 78 rats were negative. Of the 422 mice, the positive rate by culture was 7.11% (30/422), of which, 10 were S. aureus, 22 were P. aeruginosa, and 2 were K. pneumoniae. The positive rate by PCR was 7.58% (32/422), of which, 10 were S. aureus, 25 were P. aeruginosa, and 2 were K. pneumoniae. Conclusions The high sensitivity, rapid procedure and easy to operate of PCR assay makes it valuable for rapid bacteria diagnosis and large-scale screening in laboratory animals.