Objective To establish a fluorescence quantitative Taqman-PCR method for rapid and accurate detection of mouse poxvirus. Methods After sequence alignment and comparison, ERPV_027 gene was selected as the primer and probe design gene. Furthermore, the specificity, sensitivity, stability and reproducibility of these primers and probes were detected. Results The detection limitation of this method was 68 copies/μL. Data showed that this method has high specificity, which specifically amplifies mouse poxvirus, with no amplification signal of mouse hepatitis virus, Sendai virus, Salmonella and some other viruses and bacteria. This method also showed good stability and reproducibility. Conclusions This study has successfully established a fluorescence quantitative Taqman-PCR method for detection of mouse poxvirus, with high specificity, sensitivity, good stability and reproducibility, and a broad application potential.