West Nile virus (WNV), a Flavivirus distributed most widely, is presenting lately variable epidemiological and ecological patterns, including an increasing virulence that has already caused over 1000 human deaths in USA. Currently, diagnosis of WNV is achieved mainly by enzyme-linked immunoassays (ELISAs) based on the use of inactivated whole WNV (iWNV) as antigen, although results have to be confirmed by plaque reduction neutralization tests (PRNTs). Expression of WNV envelope recombinant E (rE) protein and its usefulness as ELISA antigen are described. Production of rE was achieved upon infection of Trichoplusia ni insect larvae with a recombinant baculovirus. Once optimized, the rE-based ELISA was validated with a battery of mouse and equine sera characterized previously. Concordance with the iWNV-based ELISA used routinely was good (95%), as it was with the reference PRNT (90%), with specificity of 94.4% and sensitivity of 88.1%. Production of rE protein in insect larvae allows for an easy, low cost and quite large-scale yield of partially purified antigen which is suitable for serological diagnosis of WNV, without the need for manipulation of large quantities of infective virus.