Abstract: In order to prepare monoclonal antibodies against mouse Sendai virus（SeV） HN protein, in the experiment, the SeV reference strain HN gene（GenBank accession number NC＿001552.1） was used, and the codon of the gene encoding the HN protein was optimized and then cloned and recombined into the prokaryotic expression vector pGEX-6P-1 to obtain the recombinant plasmid pGEX-6P-1-HN; it was transformed into Escherichia coli BL21 competent cells, and induced to be expressed by IPTG; the SDS-PAGE analysis was performed to purify the recombinant protein rHN. The recombinant protein rHN was used to immunize Balb/c mice for 6 times; the spleen cells were taken and fused with mouse myeloma cells SP2/0; the positive hybridoma cells were screened by subcloning and indirect ELISA, which were then injected into the peritoneal cavity of the mice to prepare the monoclonal antibodies against the HN protein; the immunoreactivity, specificity and stability of the monoclonal antibodies were analyzed. The results showed that three strains of hybridoma cells 2F9, 5D8 and 10H2 secreting anti-SeV HN protein antibodies were obtained, and the titer of the secreted antibodies was about 1∶16 000. The subtypes of these three monoclonal antibodies were all IgG1 subtypes, and all of them could specifically recognize SeV and did not cross-react with other mouse viruses with good specificity. After 10 passages, the titer of the three monoclonal antibodies could be maintained above 1∶8 000 with good stability. The results suggested that the anti-SeV HN protein monoclonal antibodies with good immunogenicity, specificity and stability had been successfully prepared, which could provide effective reagents for the detection of SeV.