To express and prepare polyclonal antibody of Human papillomavirus type 6b (HPV6b) E7 protein. a prokaryotic expression vector pGEX-4T-2/HPV6b E7 was constructed and GST-HPV6b E7 fusion protein was expressed as a soluble protein in E. coli. The expressed fusion protein was purified via Glutathione-Sepharose 4B column and thrombin cleavage in order to obtain HPV6b E7 protein. Polyclonal IgG antibody was prepared by immunizing New-Zealand rabbits with HPV6b E7 protein. Western-Blot and immunofluorescence analysis showed that the polyclonal IgG antibody could specifically recognize HPV6b E7 protein and its titer was identified. SDS-PAGE analysis demonstrated that large amounts of soluble GST-HPV6b E7 fusion protein was expressed in E. coli after 3.0-6.0 hours of IPTG induction. Polyclonal IgG antibody successfully prepared from immunized rabbits showed high titer and high specificity as confirmed by Western-Blot and immunofluorescence. The preparation of anti-HPV6b E7 polyclonal antibody will facilitate further research on the biological and immunological functions of HPV6b E7 protein.