In this research, four monoclonal antibodies (mAbs) were first generated as an immunogen by using the GST fusion protein that carries the fusion peptide and helix A derived from H7N9 influenza A virus (IAV). These mAbs could react with HA of H7N9, H3N2, and H9N2 with neutralizing activity. A novel linear epitope recognized by these mAbs was identified by peptide-based ELISA, and this epitope was located in TAADYKSTQSAIDQITGKLN at the C terminus of the helix A of H7N9. 3?A11, which is one of the four mAbs, could efficiently recognize the corresponding epitopes derived from H9, H7, H5, H3, and H1. Analysis of sera against the corresponding epitope from different HAs revealed that the C terminus of helix A in H9, H7, and H3 possessed dominant B cell epitopes that cross both Group 1 and Group 2 IAV, whereas the C terminus of helix A in H5 possessed only dominant B cell epitopes that cross subtypes in Group 1 virus. All these results demonstrated that the linear epitope identified in the helix A of H7N9 could be a novel target for developing broad-spectrum influenza diagnostics or vaccine candidates.