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Infections caused by bovine herpesvirus 1 (BoHV-1) remain a serious global issue to the health and welfare of the bovine industry. Monitoring of neutralizing antibodies is essential not only for epidemic diagnosis, but also to assess vaccination efficacy. In this study, we generated a neutralizing monoclonal antibody, termed as 3F8, targeting glycoprotein D (gD) of BoHV-1. This monoclonal antibody could neutralize BoHV-1 with a 50% inhibitory concentration (IC 50 ) of 37.82 ng/mL. Furthermore, 3F8 could inhibit BoHV-1 infection and cell-to-cell spread at the prebinding stage. A blocking enzyme-linked immunosorbent assay (ELISA) for detecting neutralizing antibodies against BoHV-1 was then developed based on 3F8 and protein gD generated using a baculovirus expression system. The sensitivity and specificity of the test were estimated to be 94.59% and 93.42%, respectively. A significant correlation (R 2  = 0.9583, p < 0.01) was observed between the results obtained with the blocking ELISA and a virus neutralization test, which suggested that the blocking ELISA could detect neutralizing antibodies against BoHV-1. A serological survey was carried out in the dairy farms in Beijing district using 3F8-based blocking ELISA to monitor the annual neutralization antibody against BoHV-1 during 2012-2020. It revealed that the dairy farms in Beijing were at high risk of BoHV-1 infection during 2012-2017 but were protected since 2018 upon implementation of an immunization program. Our results demonstrated that this assay is suitable for BoHV-1 surveillance and vaccination efficacy in cattle as a replacement for the virus neutralization test. KEY POINTS: • Prevention of BoHV-1 infection requires the monitoring of neutralizing antibodies. • A blocking ELISA for the neutralizing antibody was developed based on mAb 3F8 against BoHV-1 gD. • It can replace the labor-intensive and time-consuming viral neutralizing tests., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)