Gammaherpesviruses in the genus Macavirus establish clinically unapparent persistent infections in reservoir species. Transmission of some of these viruses, including alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2), to clinically susceptible species in the order Artiodactyla can result in malignant catarrhal fever (MCF), a usually fatal lymphoproliferative disease. Serology can be used to identify MCF virus (MCFV)-infected carrier animals. However, all current serological assays utilize AlHV-1 antigens, thus none is specific for OvHV-2. In situations where sheep and other MCFV carriers are present, such as in zoos and game farms, an OvHV-2-specific assay would determine if OvHV-2 is present in the population. In this study, a recombinant protein containing a truncated OvHV-2 Ov8 glycoprotein was expressed and evaluated as a suitable target antigen to specifically detect OvHV-2 infection using an enzyme linked immunosorbent assay (ELISA). A competitive inhibition (CI)-ELISA that detects an epitope conserved among all MCFVs was used to categorize, as positive or negative, sera from 205 domestic sheep. The Ov8 assay showed 100% diagnostic sensitivity, 98.97% diagnostic specificity, 99.07% positive predictive value, and 100% negative predictive value and very high agreement (kappa = 0.990 and 95% CI = 0.971-1.000) with the CI-ELISA. Sera from animals infected with MCFVs other than OvHV-2 did not cross-react with Ov8 (100% negative predictive value). These data support the use of the Ov8 ELISA as an OvHV-2-specific diagnostic assay.