Immunoglobulin (Ig) of Japanese flounder (Paralichthys olivaceus) was purified by a combination of salting-out and DEAE Sepharose Column chromatography. The purified immunoglobulin had an apparent molecular weight of 74 kDa (heavy chain) and 24 kDa (light chain) in SDS-PAGE. Eighteen hybridomas secreting monoclonal antibodies (MAbs) against Japanese flounder Ig were obtained by immunisation of Balb/C mice with purified Ig preparations, which were selected on the basis of the double indirect enzyme-linked immunosorbent assay (D-ELISA). Two of them designated as 2D8 and 2H1 were cloned by limiting dilution and characterised with western blotting, indirect immunofluorescence assay test (IIFAT) and fluorescence-activated cell sorter (FACS) analysis. Under reducing conditions in western blotting, both MAb 2D8 and MAb 2H1 were specific for the heavy chain of Japanese flounder Ig. MAb 2D8 was used to identify surface Ig-positive lymphocytes in the peripheral blood, spleen and pronephros of healthy Japanese flounder by flow cytometry. FACS analysis revealed that 40.48% of lymphocytes in the peripheral blood, 17.32% in the spleen and 9.67% in the pronephros were reactive to 2D8.