A ranavirus that caused high mortality of largemouth bass (Micropterus salmoides) in Huangpi, Hubei was isolated and identified by using cell culture and transmission electron microscopy (TEM). The clinical symptom of diseased fish showed hemorrhages and ulcers on the skin and a pale liver. The typical cytopathic effect (CPE) was observed in the cells of the mandarin fish brain (MFB) that were infected with the filtrate of diseased fish tissue homogenate, and the viral titer was approximately 10^8.36±0.15 TCID₅₀/mL. A large number of hexagonal virus particles with a diameter of approximately 150 nm aggregated in a pseudocrystalline array in infected MFB cells. The mortality of artificial infection reached 100% within 7 days post infection, and the clinical symptoms were similar to those of naturally diseased fish. A 241 bp specific positive fragment was obtained by a largemouth bass virus (LMBV) PCR assay of both naturally diseased fish and infected cells. According to the sequence of major capsid protein (MCP) gene of LMBV in GenBank, the specific primers were designed and a 1392 bp full length sequence of open reading frame (ORF) was obtained from the above samples. The amino acid sequence shared 100% identity with that of Santee Cooper frog virus, guppy virus type 6 and largemouth bass ulcer syndrome virus. Phylogenetic analysis showed that the virus had the highest homology with ranavirus which infects fish, such as mandarin fish ranavirus, Santee-Cooper ranavirus, largemouth bass ulcerative syndrome virus, guppy virus et al. MCP amino acid sequence analysis indicated that the virus belonged to genus Ranavirus of the family Iridoviridae, and the virus was designated as largemouth bass ranavirus LMBRaV-HB001. The typical CPE could also be observed in epithelioma papulosum cyprinid (EPC) cells, grass carp ovary (GCO) cells, giant salamander muscle (GSM) cells, and gibel carp brain (GiCB) cells infected with cell cultured virus, and the viral titer reached over 10^8.0 TCID₅₀/mL. In this study, LMBRaV was first isolated and identified in cultured largemouth bass in Hubei province, and the sensitive cell lines of the virus were also screened. These results will provide critical foundation for the transmission, diagnosis, prevention and control of ranavirus.