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This invention relates to a hybridoma cell line, its secreted mouse anti-human Hb monoclonal antibody, and its applications, belonging to the fields of immunology and in vitro diagnostics. A hybridoma cell line is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: C2023339. The hybridoma cell line prepared by this invention can stably secrete mouse anti-human Hb monoclonal antibodies. The mouse anti-human Hb monoclonal antibodies secreted by the hybridoma cell line have characteristics such as small batch-to-batch variability and high affinity. The detection kit prepared using the mouse anti-human Hb monoclonal antibody secreted by the hybridoma cell line of this invention has a wide linear range, high specificity, and good positive / negative differentiation.