The highly pathogenic avian influenza viruses (HPAIVs) of subtype H5, particularly those of the currently circulating clades 2.3.2.1 and 2.3.4.4, are largely responsible for the sporadic human infections that frequently present with a high case fatality rate. Consequently, there is an urgent necessity for the development of advanced antiviral therapeutic options against the H5 HPAIVs. Herein, the yeast two-hybrid system was employed for identifying seven nanobodies that bind the HA1 domain of hemagglutinin (HA). Among these nanobodies, Nb10 was found to exhibit high-affinity and broad-spectrum neutralization capacity against viruses of clades 2.3.2.1 and 2.3.4.4 under both