<p>Influenza A virus (IAV) is a highly infectious enveloped RNA virus that primarily causes respiratory disease. Continuous antigenic variation in influenza virus hemagglutinin (HA) is a major driver of influenza pandemics. Therefore, precise identification of B-cell epitopes within HA is essential for developing diagnostic tools and vaccine candidates that can help limit further influenza spread. In the present study, an integrated experimental and bioinformatics strategy was used. A trimeric HA protein was rationally designed to improve antigenicity and immunogenicity, expressed in a mammalian eukaryotic expression system, and purified by Ni-affinity chromatography. After animal immunization, nine HA-specific monoclonal antibodies (mAbs), namely 5D5, 6B8, 9E7, 13F8, 14C3, 15F6, 16B5, 17H7, and 20C5, were generated. These mAbs specifically recognized HA protein in western blotting and indirect immunofluorescence assays (IFA). B-cell epitopes on HA were subsequently mapped using these mAbs by indirect enzyme-linked immunosorbent assay (ELISA), dot-blot, western blotting, and IFA. Six previously unreported linear B-cell epitopes were identified: ²¹GNDNSTATL²⁹, ⁴¹IVKTITNDR⁴⁹, ¹¹⁶YDVPDYASL¹²⁴, ³⁸²DLKSTQAAI³⁹⁰, ⁴⁵⁸SEMNKLFEK⁴⁶⁶, and ⁴⁸⁸KCDNACIGS⁴⁹⁶. Bioinformatics analysis further revealed the spatial distribution and structural characteristics of these epitope regions on HA. These findings expand the current understanding of HA epitopes and provide a theoretical basis for developing IAV subunit vaccines and highly sensitive detection methods.</p>