Abstract: 【Objective】The preparation of active IgT heavy chain monoclonal antibody of tilapia provided basic material for the study of tilapia mucosal immune response.【Method】Total RNA was extracted from the spleen tissues of GIFT tilapia infected with Streptococcus agalactis. The CH2 gene fragment of IgT heavy chain constant region in tilapia was amplified via PCR, and a prokaryotic expression vector was constructed. This vector was then used to transform Escherichia coli BL21（DE3） competent cells. Recombinant protein expression was induced using isopropyl-β-D-1-thiogalactopyrano-side（IPTG）, and the protein was subsequently purified using Ni-Agarose Resin. Six-to eight-week-old BALB/c mice were immunized with the recombinant protein. Following 4 rounds of immunization, mouse splenocytes were fused with SP2/0 myeloma cells. Positive hybridoma cell lines were identified through indirect enzyme-linked immunosorbent assay（ELISA）. The monoclonal antibody subtypes, titers and specificity were characterized by indirect ELISA and Western blotting.【Result】The CH2 gene fragment of IgT heavy chain constant region was amplified to a length of approximately546 bp, and the prokaryotic expression vector pET-32a（+）-IgT was successfully constructed. The recombinant protein,with an estimated molecular weight of 38.3 kD, was obtained following induction with IPTG at a final concentration of1 mmol/L, and it was expressed as inclusion bodies. Purified recombinant protein was used to immunize mice, resulting in serum antibody titers exceeding 1∶1280000, which satisfied the requirements for cell fusion. Four positive hybridoma cell lines with stable antibody secretion was identified and 2B11 was stored at the Chinese Typical Culture Collection Center （collection number: C2023390）. Subtype identification revealed that the heavy chain of 2B11 belonged to the IgG2b isotype, and the titer of the mouse ascites antibody before purification reached 1∶16000 and was 1∶8000 after purification. Western blotting analysis confirmed that the monoclonal antibody specifically recognized the natural IgT of approximately 42 kD present in the skin mucus of tilapia. 【Conclusion】The active IgT monoclonal antibody against tilapia has been successfully developed. This antibody can be used to detect the production of antibodies in tilapia after pathogenic microbial infection or vaccination.