H3N3 avian influenza viruses (AIVs) are less prevalent in poultry than H3N8 viruses. However, although relatively rare, reassortant H3N3 viruses have been known to appear in both domestic poultry and wild birds. In this study, we isolated the H3N3 virus in chickens sourced from a live poultry market in China. A comprehensive genomic analysis revealed that the virus possessed a single basic amino acid in the cleavage site of the hemagglutinin (HA) gene. Phylogenetic analysis indicated that eight genes in the H3N3 virus belong to the Eurasian lineage. Specifically, the HA and NA genes were clustered with H3N2 and H11N3, respectively, while the internal genes were closely related to the H3N8 and H9N2 viruses. Furthermore, the H3N3 virus exhibited high and moderate stability in thermal and acidic conditions and efficient replication capabilities in mammalian cells. The H3N3 virus demonstrated that it could infect and replicate in the upper and lower respiratory tract of BALB/c mice without prior adaptation, triggering hemagglutination inhibition (HI) antibody titres ranging from 80 to 160; notably, the H3N3 virus replicated vigorously within the chicken respiratory and digestive tracts. The virus also transmitted efficiently and swiftly among chickens through direct contact, leading to higher levels of HI antibodies in both the inoculated and contact birds. These findings suggest that the H3N3 virus may be a novel reassortant originating from viruses circulating in domestic poultry, thus demonstrating an increased pathogenicity and transmissibility in chickens. Our study determines that H3N3 AIV potentially threatens the poultry industry and public health, highlighting the importance of active surveillance of AIVs.