Abstract: In this study, the TNP-specific IgM was purified from Nile tilapia (Oreochromis niloticus) by affinity chromatography and immunized the Babl/c mice for monoclonal antibody (McAb) preparation. The splenocytes from immunized mice were fused with myeloma cells (SP2/0) by hybridoma technique. The positive cells, secreting anti-tilapia IgM antibodies, were screened by enzyme-linked immunosorbent assay (ELISA), Western blotting and flow cytometry. Two hybridomas were identified, named 3B3 and 9D12, and both monoclonal antibodies were prepared. The titers of purified McAb, 3B3 and 9D12 at the concentration of 1 mg/mL, were 740,741 and 359,712 units/mL, respectively. Utilizing the McAb, confocal laser microscopy detection showed that membrane IgM (mIgM) was located on the surface of B cell membrane. Distribution analysis of mIgM+ cell in immune tissues by flow cytometry revealed its tissue-dependent, with the maximum in peripheral blood (PBL) about 37.6%, followed by the spleen (SPL) 33.7%, the anterior kidney (AK) for about 23.9%, and the posterior kidney about 20%. The phagocytosis of mIgM+ B cells uptaking fluorescent microspheres showed that the phagocytic ability of mIgM+ B cells was higher on 0.5 m beads than for 1 m beads. The phagocytosis of mIgM+ B cells varied from immune tissues, with the uptaking 0.5 m beads highest in PBL but AK occupying the maximum for 1 m beads. Taken together, the anti-Nile tilapia IgM McAb was successfully prepared, and the mIgM+ B cells had the tissue-dependent phagocytic activity, indicating that mIgM+ B cells might play an important role in innate immune response.