The emergence of novel H3N8 and H10N3 avian influenza A viruses (IAVs) circulating in chicken flocks in China has raised significant concerns to their spillovers in humans and associated economic losses. In this study, we isolated a novel H3N3 strain from chickens exhibiting respiratory symptoms, designated as A/chicken/Shandong/118/2023(H3N3) (SD118). Genetic analyses revealed that SD118 was a triple-reassortment virus, possessing hemagglutinin (HA) gene originating from H3N8, neuraminidase (NA) gene originating from H10N3, and other six internal genes were from H9N2, each of which is currently circulating in chicken flocks in China. In vitro studies confirmed that SD118 could effectively replicate in mammalian cells (MDCK and A549 cells), and exhibited dual receptor-binding affinity for both avian-like (α-2, 3 sialic acid) and human-like (α-2, 6 sialic acid) receptors. Moreover, SD118 could replicated effectively not only in the lungs and nasal turbinates of the infected mouse with more than 10 % bodyweight loss, but also in the upper respiratory tract and intestines of the infected chickens with efficient transmission ability. Our findings highlight the ongoing evolution of H3 avian IAVs through reassortment with other subtypes in poultry and the enhanced surveillance is critical to monitor the genetic diversity and zoonotic potential of H3 viruses in chickens.