Abstract Background Viral Hemorrhagic Septicemia Virus (VHSV) is a rhabdovirus that causes high mortalities linked to high economic losses in aquaculture. It has been grouped in four genotypes of which some do not easily propagate on continuous cell lines. As an alternative, the objectives of this study was to develop a primary gill epithelial cell (GEC) model from olive flounder ( Paralichthys olivaceus ) as an in vitro model for the propagation of VHSV. Results Our findings show that the primary GECs developed herein are highly permissive to replication of the JF-09 genotype IVa strain leading to high cytopathic effect observed within 96 hours post virus inoculation. Our findings also show that the viability GECs produced herein corresponded with increase in the concentration fetal bovine serum in growth medium. We envision that GECs produced herein will heighten our understanding of immune mechanisms associated with virus entry on gill mucosal surfaces in flounder.