A chimeric antigen designated B103 containing six immunodominant regions derived from three structural proteins of Rubella virus (RV) was designed and its utility in serological diagnosis was assessed. Protein B103 is comprised of aa 1–30 & aa 96–123 of C protein, aa 31–105 of E2 protein, as well as aa 11–39, aa 154–277 & aa 389–412 of E1 protein. In addition, it contains thioredoxin (TRX) at the N-terminal and His tag at the C-terminal. B103 was expressed in Escherichia coli BL21(DE3) and purified by Streamline Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B103 as verified by Western blotting analysis, we constructed and evaluated a novel capture ELISA for RV-IgM detection. B103 was expressed in a soluble form, accounting for 18.57% of the total bacterial proteins. After purification, the concentration and purity of protein B103 were 3.026 mg/mL and 95.35%, respectively. Western blotting analysis demonstrated that protein B103 could react with acute-phase serum of RV. By ELISA, 40 negative sera and 40 RV-acute phase sera were detected. The sensitivity, specificity, positive predictive value, negative predictive value and coincidence rate of the ELISA were 92.50%, 95.00%, 94.87%, 92.68% and 93.75%, respectively. The McNemer analysis suggested that there was no statistical difference between the ‘Gold standard’ and the novel ELISA with a kappa coefficient of 0.900, indicating excellent consistency. B103 chimeric protein with excellent antigenicity obtained from prokaryotic expression followed by chromatography purification could prove useful for early diagnosis of RV infection.