Abstract: The CD4-1 molecule is a key membrane protein on the surface of T lymphocytes in teleost fish, playing a crucial regulatory role in adaptive immune responses. The lack of highly specific monoclonal antibodies against fish CD4-1 has limited the study of T-cell immune function and related adaptive immunity mechanisms in fish. To prepare monoclonal antibodies (mAbs) against CD4-1 of humpback grouper (Cromileptes altivelis) (CaCD4-1), a prokaryotic expression vector for CaCD4-1 was constructed in this study. The recombinant protein rCaCD4-1 was induced, expressed, and purified. BALB/c mice were immunized with rCaCD4-1, and hybridoma technology was employed for cell fusion and screening. Seven hybridoma cell lines (6H3, 6A2, 6B2, 6E1, 6A4, 6A5, 6H2) stably secreting anti-CaCD4-1 mAbs were successfully obtained. ELISA results showed that all mAbs were of the IgG type, with 6E1 belonging to the IgG2a subclass and the others to IgG1. Ascites were produced from the 6H3 cell line, and the antibody titer was determined by ELISA to be higher than 1:1088000. Western blot analysis indicates that this antibody specifically recognizes rCaCD4-1 without cross-reacting with rCaCD4-2. Flow cytometry analysis of the antibody's reactivity to native antigens showed that the antibody specifically recognized lymphocytes in the head kidney of C. altivelis, with a CD4-1⁺ T cell population of 12.3%. These results indicate that the generated monoclonal antibody exhibits high affinity and excellent specificity, making it a valuable tool for further functional studies of CaCD4-1 and in-depth analysis of adaptive immune mechanisms in humpback grouper.