Objective To establish an indirect immunofluorescence assay for detection of murine norovirus (MNV). Methods Mouse leukaemic monocyte macrophage cell line RAW264.7 cells were infected with MNV-1 and cultured for 36 hours to collect the virus and uninfected cells, and to make antigen glass slides. BALB/c mice were gavaged with MNV-1 (10⁷TCID50) and infected sera were collected as positive control. The serum was 1:10 diluted and used for measuring MNV antibody by immunofluorescence assay (IFA). 80 serum samples were tested using the two methods, IFA and ELISA, and the discrepant samples were validated by Western blotting. Results RAW264.7 cells were infected with MNV-1 for 36-48 h, showing an infection rate of 60% of the cells, and the cells infected for 36 h were preferred. IFA method was used to detect the serum with MNV-1 infection and showed that the antibody content was gradually increased at one week after infection, reaching a maximum antibody concentration at 4 weeks after infection, and maintained a stable level later. The mouse serum at four weeks after MNV-1infection was used as positive quality control. Among the 80 serum samples, 27 positive and 53 negative cases were detected by IFA method, and 32 positive and 48 negative cases were detected by ELISA. The five discrepant samples were verified by Western blotting, resulted in 3 positive and 2 negative cases. The coincidence rate of IFA was 96.0% and that of ELISA methods was 97.5%. Conclusions Basically, immunofluorescence assay can be used to detect the MNV-1 infection in mice, although false negative result may occur occasionally. IFA and ELISA detection can be selected as initial screening measures, and use Western blot assay to verify the discrepant samples.