Objective To determine whether the polypeptide antibody induced by human papillomavirus (HPV)L1 C-terminal conserved sequence polypeptide can be used to detect HPV in cervical exfoliated cell specimens. Methods Cervical exfoliated cell specimens were collected and divided into two groups: one was subjected to nested polymerase chain reaction (Nest-PCR) for detection of HPV DNA, and the other was subjected to sandwich enzyme-linked immunosorbent assay (ELISA), in which rabbit anti-polypeptide purified antibody and mice anti-polypeptide antiserum were used to determine HPV L1 in specimens. The detection rates of HPV were compared between the two groups. Part of the specimens were used for immunocytochemistry detection. Results Nest-PCR with primer MY09/11 detected 30 HPV DNA positive specimens out of a total of 296 ones, with a detection rate of 11.15%. Then the HPV DNA negative specimens were further amplified with primer GP5+/6 , and 51 (21.34%) more positive cases were identified, making the total detection rate of Nest-PCR being 30.11% (totally 81 cases). ELISA identified 91 positive cases out of 263 specimens, with a detection rate of 34.60%, which was not significantly different from that by Nest-PCR. Immunocytochemistry detection results displayed that the polypeptide antibody could specifically reveal cervical exfoliated cells infected with HPV. Conclusion The HPV L1 polypeptide antibody can satisfactorily detect the HPV infection in cervical exfoliated cells, indicating that HPV L1 polypeptide antibody has great potential in developing HPV detection kit for cervical cancer preventive screening.