Japanese Encephalitis virus (JEV) is a mosquito-borne flavivirus with a positive-sense single-stranded RNA genome that contains a big open reading frame (ORF) flanked by 5′- and 3′- untranslated regions (UTRs). Nearly 30,000 JE cases with 10,000 deaths are still annually reported in East Asia. Although the JEV genotype III vaccine has been licensed, it elicits a lower protection against other genotypes. Moreover, no effective treatment for a JE case is developed. This study constructed a pBR322-based and cytomegaloviruses (CMV) promoter-driven JEV replicon for the production of JEV single-round infectious particles (SRIPs) in a packaging cell line expressing viral structural proteins. Genetic instability of JEV genome cDNA in the pBR322 plasmid was associated with the prokaryotic promoter at 5′ end of the JEV genome that triggers the expression of the structural proteins in E. coli. JEV structural proteins were toxic E. coli, thus the encoding region for structural proteins was replaced by a reporter gene (enhanced green fluorescent protein, EGFP) that was in-frame fused with the first eight amino acids of the C protein at N-terminus and the foot-and-mouth disease virus (FMDV) 2A peptide at C-terminus in a pBR322-based JEV-EGFP replicon. JEV-EGFP SRIPs generated from JEV-EGFP replicon-transfected packaging cells displayed the infectivity with cytopathic effect induction, self-replication of viral genomes, and the expression of EGFP and viral proteins. Moreover, the combination of JEV-EGFP SRIP plus flow cytometry was used to determine the half maximal inhibitory concentration (IC50) values of antiviral agents according to fluorescent intensity and positivity of SRIP-infected packaging cells post treatment. MJ-47, a quinazolinone derivative, significantly inhibited JEV-induced cytopathic effect, reducing the replication and expression of JEV-EGFP replicon in vitro. The IC50 value of 6.28 µM for MJ-47 against JEV was determined by the assay of JEV-EGFP SRIP infection in packaging cells plus flow cytometry that was more sensitive, effective, and efficient compared to the traditional plaque assay. Therefore, the system of JEV-EGFP SRIPs plus flow cytometry was a rapid and reliable platform for screening antiviral agents and evaluating antiviral potency.