In the present study, a new hepatic tissue‐origin cell line from European eel Anguilla anguilla has been developed and characterized. This cell line designated EL has been maintained in Leibovitz L‐15 supplemented with 10% fetal bovine serum over 72 months, and subcultured more than 90 times. The EL cell line consisted predominantly of fibroblast‐like cells, which could survive over 100 days in vitro, and could grow at 15–32°C. The optimum temperature for growth was 27°C. The chromosome analysis revealed a modal diploid karyotype of 2 n = 38. The origin of this cell line was confirmed by the 18S recombinant (r)RNA sequencing. The susceptibility test indicated significant cytopathic effects in the EL cells with regard to the Rana grylio virus and the Herpesvirus anguillae . The viral replication was confirmed by transmission electron microscopy and polymerase chain reaction analysis. Following poly (I:C) exposure, the expression levels of the immune‐related molecules interferon regulatory factor‐7 ( irf7 ) and transforming growth factor‐ β (TGF‐ β ) were downregulated in EL cells, whereas the expression levels of the rf3 and the cytochrome P450 ( CYP450 ) were upregulated. All four genes were significantly upregulated following inflammation by lipopolysaccharide ( LPS ). These data suggested the application of EL cell line for viral identification, as well as for immunodiagnosis and pharmacological targeting.