〔Abstract〕 Objective To prepare monoclonal antibodies(mAbs) against nucleocapsid protein(N) of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) and identify their biological characteristics. Methods The recombinant SARS-CoV-2 N protein was induced for expression in Escherichia coli.Based on hybridoma technology, SP2/0 mouse myeloma cells were fused with the spleen cells of BALB/c mice immunized with the recombinant protein.The positive hybridoma cell lines were screened by indirect enzyme-linked immunosorbent assay(ELISA) and limiting dilution method.The subtype of mAbs was also identified by indirect ELISA.Then, a large amount of mAbs was produced by ascites injection.After purification with Protein G affinity column, the purity, titer and specificity of mAbs were measured by SDS-PAGE,indirect ELISA and Western blot. Results A total of five hybridoma cell lines that could stably secrete SARS-CoV-2 N protein specific mAbs were obtained and named as N1 to N5.Five mAbs were collected from ascites.Their subtypes were identified as IgG2 a*κ for one mAb and IgG1*κ for the others, and their titers were above 2×104.Western blot showed that the five mAbs could bind to recombinant SARS-CoV-2 N protein. Conclusion Five mAbs that can specifically recognize recombinant SARS-CoV-2 N protein were successfully prepared by hybridoma technology, which lays a foundation for exploration of immunodiagnostic reagents.