A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against glycoprotein gE (gE) of bovine herpesvirus 1 (BHV1). The assay is based on the use of two monoclonal antibodies directed against different antigenic domains on gE. Sera from uninfected cattle and cattle that had been repeatedly vaccinated with gE-negative marker vaccines scored negative, whereas sera from cattle naturally or experimentally infected with BHV1 field strains scored positive in the gE-ELISA. Antibodies against gE appeared in the serum around 11 days after infection. Cattle that were first vaccinated and then challenged, thus having less virus replication, also became gE-seropositive. The sensitivity and specificity of the gE-ELISA is high, and therefore the gE-ELISA is suitable for differentiating between infected cattle and vaccinated cattle with a gE-negative vaccine.