AIM To construct the prokaryotic fusion expression vector of human beta defensin 4 (HBD4), and to express GST-HBD4 fusion protein in Escherichia coli (E.coli) and prepare polyclonal antibody of GST-HBD4. METHODS The gene encoding mature peptide of HBD4 (mHBD4) was amplified by PCR from cloning vector PMD18-T/HBD4 which contained the full-length HBD4 cDNA and then cloned into prokaryotic expression vector PGEX-4T-2 to construct PGEX-4T-2/mHBD4. GST-HBD4 expression was induced by IPTG. The antiserum was prepared by immunizing rabbit with GST-HBD4.The titer and specificity of the antibody were detected by ELISA and Western blot, respectively. RESULTS The recombinant expression vector PGEX-4T-2/mHBD4 was successfully constructed. After being induced by IPTG, The fusion protein with relative molecular mass of 32 000 was successfully expressed in E.coli and partly expressed in the soluble form in supernatant. The rabbit antibody against GST-HBD4 was obtained. The ELISA titer of antiserum against GST-HBD4 was about 1:128 000. Western blot analysis showed that the antiserum could bind to the expressed GST-HBD4 specifically. CONCLUSION The rabbit antibody against GST-HBD4 has been successfully prepared, which lays the foundation for further studying the structure and function of HBD4.