Objective To prepare recombinant Escherichia coli filamentous thermosensitive protein Z-interacting protein A (Ec-ZipA) 185-328 amino acid functional protein and its specific rat polyclonal antibody. Methods The synthetic Ec-ZipA gene fragment was successfully cloned into pET-30a(+) vector. After transformation, recombinant Ec-ZipA was stably expressed under an optimal bacterial expression condition in E.coli BL21(DE3) cells, and purified by HisTrap affinity chromatography. Then the biological activity of purified Ec-ZipA was further analyzed by fluorescence polarization (FP). The anti-Ec-ZipA polyclonal antibody was prepared using recombinant Ec-ZipA as antigen to vaccinate rat subcutaneously. The titer and immunological specificity of the polyclonal antibody were determined by ELISA and Western blot analysis. Results The results of SDS-PAGE and Western blot showed that recombinant Ec-ZipA was successfully expressed and purified. The FP demonstrated a perfect biological activity for purified Ec-ZipA. The titer of polyclonal antibody was 1:512000 by ELISA, and Western blot confirmed high sensitivity and specificity of polyclonal antibody against recombinant and endogenous Ec-ZipA respectively. Conclusion The prokaryotic expression, and purification of Ec-ZipA is performed, and rat anti-Ec-ZipA polyclonal antibody is successfully prepared.