Objective To establish a rapid, specific and sensitive TaqMan real-time fluorescence quantitative PCR assay for detection of murine polyomavirus (MPyV). Methods The specific primers and TaqMan probe were designed based on genome sequence of MPyV. The primers amplified a 69 bp fragment. After optimizing the reaction system and reaction condition, the standard curve was plotted by detecting recombinant plasmid standards. The specificity, sensitivity and reproducibility of this method were evaluated. In addition, samples of lungs, spleens and feces obtained from experimentally infected mice and 86 clinical samples were used to validate the efficacy of this real-time PCR assay. Results The specificity assay showed that this assay could specifically detect MPyV and the sensitivity for MPyV was about 100 copies/well. The coefficients of variation (CV) of both intra-assay and inter-assay were less than 1.13%. All of the samples from experimentally infected mice were positive for MPyV and 3 out of 86 clinical samples were positive by this TaqMan-PCR detection with a positive rate of 3.5%. Conclusions The real-time fluorescence quantitative TaqMan-PCR assay established in this study has high specificity, sensitivity and stability. It can be used for clinical diagnosis, routine detection and epidemiological investigation of murine polyomavirus infections.