Purpose To investigate the diagnostic value of p16/Ki-67 dual stain cytology for detection of cervical precancerous lesions as a novel option for cervical lesions screening.Methods A total of 295 cases diagnosed as atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LSIL) from thinprep cytologic test (TCT) were selected.Double staining of p16/Ki-67 cytology,vaginal biopsy,biopsy and pathological examination were applicated,p16/Ki-67 dual stain cytology was compared with that of biopsy and pathological examination.At the same time,The sensitivity and specificity of p16/Ki-67 dual stain cytology between ASC-US and LSIL was analyzed.Results The positive rate of p16/Ki-67 dual stain cytology were 37.42％ and 36.36％ in ASC-US and LSIL,respectively.The positive rate of cervical intraepithelial neoplasia 2/3 (CIN2/3) were 25.77％ and 25.76％.The sensitivity and specificity of the p16/Ki-67 test for detecting CIN2/3 was 83.33％ and 78.51％.The sensitivity and the specificity of the p16/Ki-67 test for detecting CIN2/3 was 85.30％ and 80.61％ in LSIL group.Conclusion p16/Ki-67 double stain cytology detection can improve the sensitivity of CIN2/3 and the specificity of human papilloma virus (HPV).p16/Ki-67 double stain detection can effectively triage the high grade cervical lesions in TCT and improve the accuracy of cervical cancer screening.