Objective To observe whether the polypeptide monoclonal antibody induced by human papillomavirus (HPV) major capsid protein (L1) C-terminal conserved sequence polypeptide can be applied to detect HPV in cervical exfoliated cells. Methods Two copies of cervical exfoliated cells specimens were collected from the gynecology clinic, The Second Clinical Medicine College, China Three Gorges University:one was subjected to detection via Human Papillomavirus Genotyping Diagnosis Kit in the clinical laboratory of hospital, and the other was subjected to detection of HPV L1 via immunohistochemistry (IHC) with the mouse monoclonal antibody induced by HPV L1 C-terminal conserved sequence polypeptide. The detection results of HPV of the two methods were analyzed. Results In a total of 384 cervical exfoliated cell samples, 63 were found positive for HPV by the Human Papillomavirus Genotyping Diagnosis Kit, with a positive rate of 16.41% (63/384); while IHC identified 120 positive cases,with a positive rate of 31.58% (120/380), being significantly higher than that of the former method (P<0.05). Conclusion It is indicated that HPV L1 polypeptide mouse monoclonal antibody has a better detection performance for HPV infection in cervical exfoliated cells compared with Human Papillomavirus Genotyping Diagnosis Kit, and it may have a future in developing kit for the prevention screening of cervical cancer.