Objectives：To explore the diagnostic value of p16/Ki-67 dual stain cytology for high-grade squamous intraepithelial lesion（HSIL）in patients with positive atypical squamous cell of undetermined significance/high risk human papillomavirus（ASCUS/HPV）. Methods：A total of 283 cases diagnosed as HSIL with positive ASCUS/HPV were selected between September，2017 and December，2018 from the Department of Gynecology，the First Affiliated Hospital of Nanjing Medical University. Colposcopy and biopsy were performed on all patients to analyze the risk factors of HSIL. And the diagnostic value of p16/Ki-67 dual stain cytology was compared with that of HPV genotype. Results：In total，88 of the 283 ASCUS/HPV positive cases were diagnosed with HSIL. Univariate logistic analysis indicated that different results of p16/Ki-67 dual stain cytology and HPV genotype test showed different HSIL rates，which were statistically significant（P＜0.01）. Ages of the patients did not contribute to the variation of HSIL incidences（P＞0.05）. Multivariable logistic analysis showed that both HPV genotype and p16/Ki-67 positives were risk factors for HSIL（P＜0.001）. The sensitivity，negative predictive value and area under ROC of p16/Ki-67 dual stain cytology were higher than those of HPV genotype test（P＜0.05）. Conclusion：p16/Ki-67 dual stain cytology positive is a warning sign of HSIL for patients with positive ASCUS/HPV，the screening efficacy of which is better than that of HPV genotype test. It provides a new option for cervical lesion screening.