Three monoclonal antibodies (MAbs) against channel catfish virus (CCV) were generated from mice immunized with purified CCV. Western blot analysis revealed that the MAb 3G12 reacted with three CCV proteins of 94 kDa, 130 kDa, and 170 kDa; the MAb 4C4 reacted with two CCV proteins of 130 kDa and 170 kDa; and the MAb 4D4 reacted with two CCV proteins of 94 kDa and 98 kDa. Indirect immunofluorescence assay showed intense fluorescence in the CCV-infected channel catfish ovary (CCO) cells in areas corresponding to the location of granular structures. In addition, the three MAbs could completely neutralize CCV at a dilution of 1:500. This study demonstrated that these MAbs could recognize CCV specifically and will be useful in the development of diagnostic methods for the detection of fish CCV infection.