Objective To investigate the expression of microRNA-508-3p (miR-508-3p) and Rho-associated protein kinase 1 (ROCK1) in exfoliated cervical cells and their diagnostic values in cervical cancer.Methods From February 2016 to September 2019, 172 cases of exfoliated cervical cells were collected in our hospital, including 70 cases in the normal group, 54 cases in the cervical intraepithelial neoplasia group, and 48 cases in the cervical cancer group. The expression of miR-508-3p and ROCK1 in each group was detected by qRT-PCR, immunohistochemistry, and Western blotting. Luciferase assay was used to verify the interaction between miR-508-3p and ROCK1 in the exfoliated cervical cells. The relationship between the expression of miR-508-3p and ROCK1 in the exfoliated cervical cells and the clinical data of cervical cancer patients were analyzed, and the diagnostic value of miR-508-3p and ROCK1 expression in the exfoliated cervical cells was evaluated via receiver operating characteristic (ROC) curve.Results Compared with the normal group, the content of miR-508-3p in cervical cancer group (P < 0.05) and cervical intraepithelial neoplasia group (P < 0.05) was decreased, while ROCK1 mRNA level, the positive rate of ROCK1 mRNA and ROCK1 protein content were increased (P < 0.05). Compared with the cervical intraepithelial neoplasia group, the content of miR-508-3p in the cervical cancer group was decreased (P < 0.05), while ROCK1 mRNA level, the positive rate of ROCK1 mRNA and ROCK1 protein content were increased (P < 0.05). MiR-508-3p was negatively correlated with ROCK1 in the cervical cancer group (r = -0.6678, P = 0.000) and the cervical intraepithelial neoplasia group (r =-0.5234, P = 0.000), and ROCK1 was the direct target gene of miR-508-3p. The relative expression levels of miR-508-3p and ROCK1 were correlated with FIGO stage and lymph node metastasis of cervical cancer (P < 0.05), but not with age and menopausal status of patients, or diameter and pathological type of tumor (P > 0.05). In the diagnosis of cervical cancer and cervical intraepithelial neoplasia, the area under the ROC curve (AUC) of miR-508-3p was 0.946 (95% CI: 0.899, 0.993) and 0.851 (95% CI: 0.782, 0.921), the sensitivity was 87.1% (95% CI: 0.776, 1.654) and 84.3% (0.746, 1.589), and the specificity was 100.0% (1.000, 2.000) and 79.6% (95% CI: 0.702, 1.497), respectively. The AUC of ROCK1 was 0.949 (95% CI: 0.892, 1.000) and 0.905 (95% CI: 0.852, 0.958), the sensitivity was 89.6% (95% CI: 0.810, 1.706) and 77.8% (95% CI: 0.667, 1.445), and specificity was 100.0% (95% CI: 1.000, 2.000) and 88.6% (95% CI: 0.812, 1.698), respectively. After 1-year follow-up, 42 cases survived and 6 cases died in 48 cases of cervical cancer. The expression level of miR-508-3p in the survival group was higher than that in the death group, and the expression level of ROCK1 mRNA in the survival group was lower than that in the death group (P < 0.05).Conclusions The expression of miR-508-3p and ROCK1 in exfoliated cervical cells of patients with cervical cancer is severely abnormal, in which miR-508-3p is downregulated while ROCK1 is upregulated. The miR-508-3p targets ROCK1 and negatively regulates ROCK1, and both of them are closely associated with the pathological staging and lymph node metastasis in cervical cancer patients. Besides, they are of diagnostic value and may be potential biomarkers for cervical cancer screening.