Abstract: This study aims to develop a differential assay for popularizing the use of E2-based marker vaccines against classical swine fever (CSF). Based on the recombinant E^rns produced by E. coli, an indirect ELISA (E^rns-ELISA) was established and optimized using the E^rns fusion protein as coating antigens. The optimal amount of coating antigen was optimized to be 0.15 μg, and the serum dilution was optimized to be 1∶100. The established E^rns-ELISA was shown to be sensitive, specific and reproducible, and has a good agreement with E2-ELISA based on the recombinant fusion E2 protein (96.7%) and Dot-ELISA based on whole viral antigens (77.5%). The E^rns-ELISA combined with E2-ELISA can be used as differential diagnostic assays accompanying marker or DIVA vaccines against CSF to differentiate animals infected with wild-type viruses from those vaccinated with E2-based marker vaccines.