Continued influenza surveillance remains important, especially given that the emergence of novel subtypes or reassorted influenza viruses with pandemic potential continues to be a worldwide threat. In particular, virus circulating in birds can facilitate interspecies transmission to humans. In this study, we conducted systematic surveillance of H3 subtype avian influenza virus (AIVs) in domestic poultry and wild birds throughout Guangdong Province from 2023 to 2025. A total of 21 strains of H3 subtype AIVs were isolated, and phylogenetic analyses and risk assessment of their internal gene segments revealed genetic evidence of reassortment events, indicating a close genetic relationship with highly pathogenic avian influenza viruses (HPAIVs). ZJ1722, ZJ1542 and SZ837 showed dual-receptor binding ability and robust replication in mammalian cells, which coincided with amino acid mutations in the HA protein associated with human receptor binding. Although the H3NX viruses isolated in this study failed to cause lethality in mice, they efficiently replicated in the nasal turbinate and lungs of mice without prior adaptation. This study highlights the paramount importance of sustained, subtype-specific surveillance targeting H3NX avian influenza viruses coupled with timely risk characterization and assessment. Proactive containment of H3NX avian influenza virus (AIV) transmission has vital implications for safeguarding the sustainability of the poultry industry and protecting global human public health, given the inherent zoonotic potential and evolutionary plasticity of this H3 subtype, which could drive future spillover events.