In this work, the two positive hybridomas designated as 1B2 and 2G9 secreting monoclonal antibodies (MAbs) against half-smooth tongue sole IgM were produced and then cloned by limited dilution. Western blotting showed that MAbs 1B2 and 2G9 could specifically react with the heavy and light chains of IgM, respectively. The indirect immunofluorescence assay (IFA) showed that green fluorescent signals were detected on the membrane of lymphocytes, which indicated that membrane-bound IgM (mIgM) could be recognized by both MAbs 1B2 and 2G9. Specificity analysis showed that no cross-reactions were detected between IgM of half-smooth tongue sole and other five teleosts IgMs using MAbs 1B2 and 2G9. Flow cytometric analysis (FCA) demonstrated that the percentages of mIgM+ lymphocytes in peripheral blood, spleen and pronephros of health half-smooth tongue sole were 21.1%, 17.6% and 16.1%, respectively. Moreover, the phagocytosis rates of fluorescence microspheres by mIgM+ lymphocytes in isolated leukocytes from peripheral blood, spleen and pronephros of health half-smooth tongue sole were determined to be 4.6 ± 0.5%, 3.6 ± 0.4% and 4.2 ± 0.3% by FCA, respectively, and the proportions of mIgM+ phagocytic lymphocytes in total mIgM+ lymphocytes in the peripheral blood, spleen and pronephros were calculated to be 21.6 ± 1.1%, 20.6 ± 1.8% and 26.0 ± 0.8%, respectively. These results demonstrated that the MAbs produced in this study could specifically recognize the serum IgM and mIgM on B lymphocytes of half-smooth tongue sole. Meanwhile, partial mIgM+ lymphocytes have the phagocytic capacity, which indicates mIgM+ lymphocyte play important role in innate immune response.