Objective To clone proline-glutamate 8 (PE8) gene segment from Mycobacterium tuberculosis (H37Rv), construct the recombinant plasmid pET28a-PE8, express recombinant PE8 protein, and prepare its polyclonal antibody. Methods Using a standard homologous recombination cloning technology, we cloned the PE8 gene into the prokaryotic vector pET28a. After sequence confirmation, it was transformed into E. coli BL21 (DE3) and treated with 0.5 mmol/L isopropyl-beta-D-thiogalactopyranoside (IPTG) to induce protein expression. We purified and renatured the recombinant PE8 protein, and immunized New Zealand rabbits to prepare the polyclonal antibody. Antibody titer was determined by indirect ELISA and the specificity was evaluated by Western blot analysis. Results The recombinant plasmid pET28a-PE8 was successfully constructed, and the PE8 protein was primarily expressed in an inclusion body in E. coli. After renaturation and purification, a purity of about 90% of the recombinant protein was achieved. The titer of the polyclonal antibody was higher than 1:430 080. The polyclonal antibody could specifically recognize the recombinant PE8 protein. Conclusion We have successfully expressed and purified recombinant PE8 protein, which can be further utilized to generate PE8 polyclonal antibody with acceptable titer and specificity.