Objective We established a rapid detection method of Pasteurella spp. and provided a reference for microbiological quality control of laboratory animal. Methods According to the β subunit of bacterial RNA polymerase (rpoB) protein multiple alignments of 13 different Pasteurella spp. published in NCBI. The degenerate primers were designed by CODEHOP designer online. CODEHOP PCR method was applied to detecting Pasteurella spp. after the specificity and sensitivity of the method had been evaluated by 21 reference strains. Results Standard strain amplified fragment were about 200 bp by degenerate primers PastF6/PastR5. The primers are able to distinguish between Pasteurella spp. and the other pathognic organisms of laboratory animal respiratory tracts. Sensitivity of this method were 0.2 pg/μL~2 pg/μL to different Pasteurella. The Pasteurella positive rate was 19.1% in 609 animal's respiratory samples. The accuracy of positive results was 100% through verifying by sequenced and blast. Conclusions The established method has good specificity and sensitivity. It can be used to detect Pasteurella spp. in animal samples.