Objective To detect the expression of p16, mini-chromosome maintenance protein 2 (MCM2) and Ki-67 in cervical squamous intraepithelial lesion (SIL) tissue, and analyze the relationship between the positive expressions among the three, provide basis for clinical diagnosis and treatment of diseases. Methods Retrospectively analysed of the clinical datas of the 240 cases patients with cervical intraepithelial neoplasia (CIN) (130 patients with CIN Ⅰ grade, 110 patients with CIN Ⅱ?~Ⅲgrade), 50 patients with chronic cervicitis and 20 patients with invasive squamous cell carcinoma of cervix, who admitted to Ji'an Maternity and Child Health Hospital from July 2017 to October 2019. The cervical biopsy tissues of each patient were collected and subjected to SP immunohistochemical staining, the expression of p16, MCM2, and Ki-67 in the cervical pathological tissues of each patient were detected, and analyze the correlation of the positive expression of p16, MCM2, and Ki-67. Results The positive expression rates of p16, MCM2, and Ki-67 in the pathological tissues of patients with CIN grade Ⅰ, CIN grade Ⅱ?~Ⅲ, and invasive squamous cell carcinoma of cervix were significantly higher than those of chronic cervix inflammation patients, and patients with CIN grade Ⅱ?~ Ⅲ , invasive squamous cell carcinoma of cervix were significantly higher than patients with CIN grade Ⅰ ; Spearman correlation analysis showed that, the positive expression of p16 and MCM2 were positively correlated (r =0.691); the positive expression of p16 and Ki-67 were positively correlated (r =0.556); the positive expression of MCM2 and Ki-67 were positively correlated (r =0.676) (all P <0.05). Conclusion p16, MCM2, and Ki-67 in patients with chronic cervicitis, CIN patients and patients with invasive squamous cell carcinoma of cervix are all abnormally highly expressed, and the positive expression of p16, MCM2, and Ki-67 positively correlated with the severity of the disease, p16, MCM2, and Ki-67 positive expression also has a significant positive correlation, which can be used as the molecular markers for SIL diagnosis.