Abstract: The study was aimed at preparing and purifying polyclonal antibody of rabbit anti-pig ANNXA2 by GST-ANNXA2-coupled affinity chromatography. The fragment of pig Annexin A2 and EGFP fusion gene was amplified from pA2EGFP plasmid by PCR. And then the fragment was subcloned into the prokaryotic expression vector pET-30a(+) to construct the recombinant plasmid p30a/AE, and expressed in inclusion body of E. coli. Rosetta (DE3) by IPTG induction. The fusion protein ANNXA2-EGFP which was purified by NiResin HP affinity chromatography was used as immunogen to inject male rabbit to prepare polyclonal antibody anti-pig Annexin A2. The soluble fusion protein GST-ANNXA2 which has been prepared was coupled on NHS-activated Sepharose Fast Flow column, and then the polyclonal antibody was purified by this column- Purified antiserum titer amounted to 1:12 800 by indirect ELISA, and the purified antiserum was also demonstrated high specificity by Western blot and immunohistochemistry staining. The polyclonal antibody of rabbit anti-pig ANNXA2 was prepared and possessed high titer and high specificity, which laid the groundwork for the investigation of relationship between virus infection and pig Annexin A2.