The first documented avian influenza virus subtype H16N3 was isolated in 1975 and is currently detectable in many countries worldwide. However, the prevalence, biological characteristics and threat to humans of the avian influenza virus H16N3 subtype in China remain poorly understood. We performed avian influenza surveillance in major wild bird gatherings across the country from 2017 to 2019, resulting in the isolation of two H16N3 subtype influenza viruses. Phylogenetic analysis showed these viruses belong to the Eurasian lineage, and both viruses presented the characteristics of inter-species reassortment. In addition, the two viruses exhibited limited growth capacity in MDCK and A549 cells. Receptor-binding assays indicated that the two H16N3 viruses presented dual receptor-binding profiles, being able to bind to both human and avian-type receptors, where GBHG/NX/2/2018(H16N3) preferentially bound the avian-type receptor, while GBHG/NX/1/2018(H16N3) showed greater binding to the human-type receptor, even the mice virulence data showed the negative results. Segments from other species have been introduced into the H16N3 avian influenza virus, which may alter its pathogenicity and host tropism, potentially posing a threat to animal and human health in the future. Consequently, it is necessary to increase monitoring of the emergence and spread of avian influenza subtype H16N3 in wild birds.