Objective To develop a rapid, specific and sensitive fluorescence real-time RT-PCR assay for detection of murine norovirus (MNV). Methods A pair of primers and a Taqman probe were designed targeting highly conserved sequences among MNV strains,which are located at the open reading frames 1 (ORF1)-ORF2 junction region. The internal control(IC)was also designed and constructed to determine false-negative results. A real-time RT-PCR assay with the IC was established for MNV detection by optimizing reaction components and conditions. The standard curve was plotted based on the linear relationship between the amount of plasmid DNA and cycle threshold(Ct)values. In addition,the specificity, sensitivity and reproducibility of the assay were evaluated. 344 clinical samples were used to determine the efficacy of this real-time RT-PCR method. Results The specificity test showed that this real-time RT-PCR assay could specifically detect MNV and had no cross reactions with mouse hepatitis virus, Theiler's murine encephalomyelitis virus, Sendai virus, pneumonia virus of mice, reovirus III, Hantaan virus and lymphocytic choriomeningitis virus. The standard curve showed fine linear relationship between the amount of plasmid DNA and Ct values (correlation coefficient r²=0.9986). The developed assay was sensitive enough to detect as low as 10 copies/μL. The lower quantification limit of MNV was estimated as 1.78×10^-2 TCID₅₀/mL,which was 10 times more sensitive than conventional RT-PCR method, and 100 times more sensitive than virus isolation method. Both the intra-batch and inter-batch coefficients of variation were less than 2% based on 5 repeated intra-batch and inter-batch test of 5 samples. 344 clinical samples were tested by this developed real-time RT-PCR assay and showed a positive ratio of 29.94% (103/344). Conclusions This real-time RT-PCR assay is a specific, sensitive and reproducible assay. Moreover,the internal control in the real-time RT-PCR system can be used to determine false negative results. This assay could be exploited for routine detection, clinical diagnosis and epidemiological survey of mouse norovirus.