Abstract A new marine fish cell line, derived from the heart of giant grouper, E pinephelus lanceolatus ( B loch), was established and characterized. The cell line was designated as ELGH and subcultured with more than 60 passages. The ELGH cells were mainly composed of fibroblast‐like cells and multiplied well in L eibovitz's L ‐15 medium supplemented with 10% foetal bovine serum (FBS) at 28 °C. Chromosome analysis indicated that the modal chromosome number was 48. The fluorescent signals were detected in ELGH when transfected with green fluorescent protein reporter plasmids. The 50% cytotoxic concentration ( CC 50 ) of the extracellular products ( ECP s) from S treptococcus iniae and V ibrio alginolyticus E 333 on ELGH cells was 60.02 and 12.49 μg mL −1 , respectively. Moreover, the ELGH cells showed susceptibility to S ingapore grouper iridovirus ( SGIV ), but not to soft‐shelled turtle iridovirus ( STIV ), red‐spotted grouper nervous necrosis virus ( RGNNV ) and spring viremia of carp virus ( SVCV ), which was demonstrated by the presence of a severe cytopathic effect ( CPE ) and increased viral titres. In addition, electron microscopy observation showed that abundant virus particles were present in the infected cells. Taken together, our data above provided the potential utility of ELGH cells for transgenic and genetic manipulation, as well as cytotoxicity testing and virus pathogenesis.