Influenza A virus causes numerous deaths and infections worldwide annually. Therefore, we have considered nanobodies as a potential treatment for patients with severe cases of influenza. We developed a nanobody that was expected to have protective efficacy against the A/California/04/2009 (CA/04; pandemic 2009 flu strain) and evaluated its therapeutic efficacy against CA/04 in mice experiments. This nanobody was derived from the immunization of the alpaca, and the inactivated CA/04 virus was used as an immunogen. We successfully generated a nanobody library through bio-panning, phage ELISA, and Bio-layer interferometry. Moreover, we confirmed that administering nanobodies after lethal doses of CA/04 reduced viral replication in the lungs and influenza-induced clinical signs in mice. These research findings will help to develop nanobodies as viral therapeutics for CA/04 and other infectious viruses. Display omitted •Through phage display technology using immunized Alpaca, H1 Nb-hIgG1 was developed.•H1 Nb-hIgG1 neutralize pandemic 2009 flu by binding to receptor binding domain.•Therapeutic efficacy of H1-Nb hIgG1 was confirmed in pandemic 2009 flu-infected mice.