Immunoglobulin M (IgM) was purified from olive flounder Paralichthys olivaceus sera using mannan-binding protein (MBP) and protein L affinity columns (designated as MBPIgM and ProLIgM, respectively). A monoclonal antibody (MAb) against olive flounder IgM was produced. The MBPIgM and ProLIgM had apparent molecular weights of 77, 73, and 28 kDa in SDS-PAGE. Nine hybridomas secreting MAbs against olive flounder IgM were established: five MAbs for MBPIgM (1, 2, 3, 4, and 5) and four for ProLIgM (6, 7, 8, and 9). Western blotting indicated that seven MAbs recognized heavy (H; MAbs 1, 2, 3, 4, 5, 6, and 7) chains and one recognized light (L; MAb 9) chains of IgM, while MAb 8 did not recognize IgM. The results of enzyme-linked immunosorbent assay (ELISA) with bovine serum albumin (BSA, antigen) and the nine MAbs revealed that the optical density (OD) values of sera differed significantly between BSA- and non-immunized fish, despite some sera from non-immunized fish with slight high OD values. These results suggest that the MAbs produced in this study reacted specifically with the IgM from olive flounder.