Objective To establish a real-time quantitative PCR (qPCR) method for detection of Streptobacillus moniliformis, which can be used to rapidly detect this pathogen in laboratory animals. Method According to the S. moniliformis sequences published in NCBI, we designed specific primers and MGB probe. The specificity, sensitivity and stability of this method were evaluated using 24 standard reference strains. Total of 823 respiratory specimens of animals including mice, rats, guinea pigs, hamsters, rabbits, Mongolian gerbils and tree shrews, were detected by this established Taqman MGB qPCR method. Results We had successfully established the S. moniliformis Taqman MGB qPCR method. S. moniliformis was not detected in the samples of mice, rats, guinea pigs, hamsters and rabbits. The positive rate of S. moniliformis was 1.5% (1/65) and 61.7% (37/60) in conventional Mongolian Gerbils and tree shrews, respectively. Conclusions Our developed qPCR method can be used to effectively detect S. moniliformis in laboratory animals. Moreover, its accuracy and sensitivity are better than the national standard method. This study laid the foundations for optimizing the quality inspection system of laboratory animals.