Infectious spleen and kidney necrosis virus (ISKNV) causes a disease with high mortality, resulting in significant economic loss to Siniperca chuatsi culture industry in China. To establish ELISA assay to determine virus antigen content of ISKNV vaccine and investigate the pathogenic mechanism of ISKNV major capsid protein (MCP), the monoclonal antibody (MAb) against recombinant MCP protein was developed and the properties were identified. The purified recombinant MCP was injected into BALB/c mice through subcutaneous route for three times. Then myeloma cells SP2/0 were fused with the spleen cells of the immunized BALB/c mice. Three hybridoma cell lines against ISKNV MCP were screened using indirect ELISA and were identified to be IgG1 subtype, designated as 5F1, 3D9 and 5B4, respectively. The three McAbs had no reaction with SCRV and STIV except ISKNV by the indirect ELISA. The hybridoma cell line 5F1 was selected for ascites preparation. When the recombinant MCP and ISKNV supernatant was used as detective antigen respectively, the titer of ascites was 1:51 200 and 1:400 respectively. Indirect immunofluorescence and Western-blot analysis showed that the McAb 5F1 could recognize authentic MCP protein of ISKNV and working concentration of McAb 5F1 was confirmed. From above, the MAb against MCP of ISKNV was successfully prepared, which laid a foundation for further study.