Our previous studies found that the H1-50 monoclonal antibody (mAb) of influenza A virus hemagglutinin (HA) cross-reacted with pancreatic tissue and islet β-cells, and further studies showed that H1-50 mAb binds to prohibitin (PHB) protein of islet β-cells. These suggest that there are heterophilic epitopes between influenza virus HA and pancreatic tissue, which may be involved in the pathogenesis of type 1 diabetes. To further investigate these heterophilic epitopes, we screened binding epitopes of H1-50 mAb using a phage 12-peptide library. DNA sequencing and comparative analysis were performed on specific positive phage clones, and the sequence of 12-peptide binding to H1-50 mAb was obtained. The binding epitopes of H1-50 mAb in influenza virus HA were determined by sequence analysis and experimental verification, and their distribution within the three-dimensional structure was assessed by PyMOL. The results showed that H1-50 mAb specifically binds to polypeptides (306-SLPFQNIHPITIGK-319) of influenza A virus HA, located in the stem of the HA protein. However, there is no specific binding sequence between H1-50 mAb and the PHB protein of islet β-cells in the primary structure, and we speculate that the binding of H1-50 mAb to islet β-cells may depend on the spatial conformation. The identification of the heterophilic epitopes of H1N1 influenza virus hemagglutinin provides a new perspective on type 1 diabetes that may be caused by influenza virus infection, which may contribute to the prevention and control of influenza.