H6 subtype avian influenza viruses (AIVs) have a broader host range and circulate globally in wild birds, domestic ducks, geese, and terrestrial poultry all over the world. Their demonstrated capacity to adapt receptor-binding affinity for mammalian species constitutes a persistent concern for zoonotic transmission and public health. In this study, a novel triple-reassortant H6N8 AIV strain was isolated from a chicken farm in southern China and designated as A/chicken/Guangdong/JM642/2023 (H6N8). The complete genome of the virus was sequenced using Next-Generation Sequencing. Phylogenetic analysis indicated that the HA gene of the isolate clustered into the Group III/HN573-like, which encompasses H6 subtype viruses bearing various NA genes and belonging to the Eurasian gene pool. The NA gene showed the closest genetic relationship with viruses originating from North America. All six internal genes were derived from H9N2 AIVs. The virus possesses several key molecular determinants known to contribute to an expanded host range and increased virulence. Animal infection studies demonstrated that the virus was capable of infecting mice without prior adaptation. It replicated efficiently in the lungs and nasal turbinates, followed by systemic dissemination resulting in lethal outcomes. Inoculated chickens remained asymptomatic; however, the virus replicated efficiently in multiple organs, with high viral loads detected particularly in the lungs and kidneys. Viral shedding occurred via both the respiratory and digestive tracts, and horizontal transmission was observed among chickens. Notably, infected and contacted chickens developed high levels of antibodies from 8 days post-inoculation (DPI) to the end of observation period. This study enhances our understanding of the genetic and biological characteristics of the novel reassortant H6N8 AIVs and underscores their potential risk to public health.