Since 2022, three cases of human infections of novel H3N8 avian influenza viruses (AIVs) have been confirmed in China. Given the potential for significant public health implications, the prompt detection and containment of the virus is particularly important. Comprehensive analyses were conducted of the complete viral gene sequences of five H3 subtype AIVs that were isolated from chickens, pigeons, and geese in live poultry markets in China in 2023. Four strains exhibited a high degree of homology with the H3N8 viruses responsible for human infections in 2022 and 2023. A subsequent study was conducted to investigate the pathogenicity differences among multiple subtypes of the H3 AIVs in chickens. The study revealed that all infected chickens exhibited clinical signs and viral shedding. Notably, two H3N8 viruses, which were highly homologous to human strains, demonstrated significant differences in adaptability to chickens. The goose-derived H3N5 strain displayed high adaptability to chickens and could replicate in multiple organs, with the highest titer in the cloaca. Additionally, a potential vaccine strain, designated CK/NT308/H3N3, was successfully developed that provided complete clinical protection and effectively prevented viral shedding against both H3N3 and H3N8 viruses. In conclusion, CK/NT308/H3N3 presents a promising vaccine candidate.