Objective To establish a real-time fluorescent quantitative PCR (FQ-PCR) method for detection of murine norovirus (MNV) in laboratory mouse and provide the basis for establishment of a standard detection method for MNV. Methods Specific primers were designed and MNV DNA standards were prepared according to the MNV genome sequences published on NCBI. The specificity, sensitivity, repeatability and stability of the established Q-PCR method were tested. The established Q-PCR method was applied to detect 766 mouse caecum content samples to explore preliminarily the infection status of laboratory mice in Beijing. Results No cross reaction showed in human norovirus and feline calicivirus with the established Q-PCR method.The sensitivity was up to 10 copies/μL. The coefficient of variation (CV) of intra-assay and inter-assay was less than 2%. There were 301 positive cases detected in the 766 samples of laboratory mice. Conclusions The established FQ-PCR method is accurate and effective with high specificity, sensitivity and repeatabiliy in the quantitative detetion of nucleic acid, and can be applied to rapidly and quantitatively screen MNV in laboratory mice.