Tembusu virus (TMUV) has been a causative agent of an acute egg-drop syndrome found in Chinese duck populations since at least 2010. In this paper, we report the characterization of a TMUV-like flavivirus (named CJD05) isolated from naturally infected egg-laying fowl. The virus was identified and then isolated from hens suffering from severe egg drop and fever in Fujian Province, China. The virus replicated well in MDEF and CEF cells, and its cytopathogenic effect (CPE) was apparent. Hemagglutinating activity (HA) was negative for this virus using erythrocytes from both chickens and pigeons. Viral particles were enveloped and approximately 45 nm in diameter, as observed by electron microscopy. Phylogenetic analysis of the full-length nucleotide sequence of CJD05 indicated that this virus is closely related to the duck-origin TMUV, belonging to Ntaya group of flavivirus. Most importantly, pathogenicity studies showed that CJD05 is highly virulent in 1-day-old chicks, 1-day-old Muscovy ducks, egg-laying chickens and shelducks. Our research highlights the increase in epidemic disease caused by avian TMUV, and subsequent outbreaks are becoming more complicated to treat. The pathogenic mechanisms of the virus are still not fully understood, further research is needed.