Objective To prepare the fusion protein MVF-ErbB3II composed of measles virus fusion (MVF) protein 288 to 302 amino acid peptide and human epidermal growth factor receptor 3 (ErbB3) 236 to 308 amino acid (ErbB3II) peptide, then prepare and characterize the anti-MVF-ErbB3II polyclonal antibody (pcAb). Methods The MVF-ErbB3II gene was synthesized artificially and subcloned into pET-21b plasmid using DNA ligase. After transformation, the recombinant MVF-ErbB3II protein was expressed in E. coli BL21 (DE3) and purified using nickel ion affinity chromatography. Subsequently, the purified MVF-ErbB3II protein was used as antigen to immunize rats subcutaneously for induction of anti-MVF-ErbB3IIpcAb. The titer of anti-MVF-ErbB3II pcAb was analyzed by ELISA. The ErbB3 specificity and targeting ability of pcAb were evaluated by Western blotting, immunoprecipitation (IP) and flow cytometry (FCM). Results SDS-PAGE confirmed that MVF-ErbB3II protein was successfully expressed and purified. ELISA showed that the titer of pcAb was 1 024 000. Western blotting, IP and FCM assays showed that the anti-MVF-ErbB3II pcAb not only had good antigen specificity against purified MVF-ErbB3II and native ErbB3 but targeted the ErbB3 dimerization interface. Conclusion The prokaryotic expression and purification of MVF-ErbB3II is successfully achieved, rat anti-MVF-ErbB3II pcAb is prepared and characterized successfully.