Liquid-based thin layer cytology (TCT) and HR-HPV detection are the most important screening methods for cervical cancer. These two methods have limited sensitivity and specificity, so some cervical lesions are still missed or misdiagnosed. This paper mainly discusses the value of P16 protein detection in cervical cancer screening. In particular, it is effective and practical in high-grade squamous epithelial and above cervical lesions (CINII+). In this retrospective study, the diagnostic specificity and positive predictive value (PPV) of P16 protein detection for cervical CINII+ lesions were significantly higher than that of TCT and HR-HPV detection, and the accuracy was the highest. P16 protein detection can also reduce the rate of missed diagnoses in HR-HPV-negative patients and reduce unnecessary colposcopic biopsies. Our data highlight the feasibility and significance of P16 protein detection in cervical disease screening.