In this study, an enzyme-linked immunosorbent assay (ELISA) using glycoprotein and a monoclonal antibody (MAb) was developed for the detection of antibodies to vesicular stomatitis virus (VSV) serotype New Jersey (NJ). The glycoprotein to be used as a diagnostic antigen was extracted from partially purified VSV-NJ, and a neutralizing MAb specific to VSV-NJ was incorporated to compete with antibodies in a blocking ELISA using glycoprotein (GP ELISA). The cutoff of the GP ELISA was set at 40% inhibition, which corresponded to a virus neutralization test (VNT) titer of 32. With this threshold, the GP ELISA exhibited 99.6% specificity for naïve sera (n = 3,005) from cattle (n = 1,040), pigs (n = 1,120), and horses (n = 845) from domestic farms. The GP ELISA did not cross-react with sera positive for foot-and-mouth disease virus, swine vesicular disease virus, or VSV serotype Indiana. The GP ELISA was more compatible with the VNT than was the nucleocapsid-based ELISA for VSV-NJ-positive sera (n = 19). Taken together, this GP ELISA could be a useful tool as an alternative to the VNT for detecting antibodies specific to VSV-NJ.