Seasonal Influenza viruses, owing to their continued evolution and high level of contagion, present a significant threat to public health around world each year. Vaccination remains the most effective strategy for preventing complications of influenza virus infection, particularly for vulnerable populations such as elderly individuals, children, and individuals with underlying health conditions. In this study, we described the de novo design for the discovery of affinity ligands targeting the conserved receptor binding site (RBS) of the influenza virus hemagglutinin (HA). Based on three-round of molecular docking, three candidate peptides, pep1, pep3 and pep4, with top-rankings were identified. Molecular dynamic simulation and per-residue decomposition further revealed the different binding mechanisms of the three peptides with HA and the key residue's contribution to the binding. The result of microscale thermophoresis indicated that the three peptides had broad-spectrum affinity for various influenza A strains and, among them, pep1 had the highest binding affinity for HA (Kd = 0.58-0.73 μmol/L). By coupling pep1 onto Sepharose gels, the affinity gel was applied to the evaluation of the chromatographic performance in the purification of HA and influenza A vaccine from mimic egg- and mammalian-based feedstocks. A recovery of 68.3 %-72.2 % at the purity of 95.9 %-97.2 % was obtained in vaccine purification, demonstrating the excellent feature of the peptide ligand. This work provided new insight into the rational design of broad-spectrum affinity peptide targeting HA and the result has potential application in the production of influenza vaccines.