Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted by Culex mosquitoes and is the leading cause of viral encephalitis in humans. JEV infection of swine, which are the main amplifying hosts for JEV, can cause reproductive failure in sows; in boars it can cause testitis and infertility. The prevalence of JEV in swine is a continuous threat to human health. A practical diagnostic method for monitoring JEV infection in swine herds is essential for control of the disease in both swine and humans. Here, we have identified a high-affinity anti-JEV NS1 monoclonal antibody (mAb) by indirect ELISA and utilized it for the development of a blocking ELISA (bELISA). The optimal NS1 protein coating concentration (2 μg/mL) and mAb working concentration (1 μg/mL) were determined by checkerboard titration. One hundred ten JEV-antibody-negative serum samples were used to establish 34.03% inhibition as the cutoff value for a negative result. By the bELISA, seroconversion in 80% of newly JEV-vaccinated pigs was detected by 7 days post-immunization, while by the commercial envelope-protein-based iELISA, seroconversion was detected in 20% of the newly vaccinated pigs. We found 98.7% agreement between the bELISA and the commercial iELISA when we tested 157 field samples using both methods. From an epidemiological survey of swine serum collected between 2014 and 2016, we found that the average JEV seropositive rate in unvaccinated commodity pigs was 8.1%, and in vaccinated boars and sows, it was 67.6%.