Objective: To explore the relationship between vaginal microecological indicators, 25-hydroxyvitamin D [25-(OH)D], vitamin B12 (VitB12) and cervical intraepithelial neoplasia (CIN). Methods: The clinical data of 108 patients with suspected CIN admitted to the hospital from June 2022 to June 2025 were retrospectively analyzed. According to the results of cervical histopathological examination, the patients were divided into CIN group and non-CIN group. The vaginal microecological indicators [Lactobacillus ratio (LB%), Nugent score, Shannon-Wiener diversity index (H')], serum 25-(OH)D and serum VitB12 were compared between the two groups of patients at admission. The related indicators of disease severity (Ki-67 proliferation index, lesion epithelial thickness ratio) in CIN group were statistically analyzed. Receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficiency of vaginal microecological indicators and 25-(OH)D and VitB12 levels at admission on CIN. Pearson correlation coefficient was used to analyze the correlation between vaginal microecological indicators, 25-(OH)D and VitB12 levels at admission and disease severity related indicators in CIN group. Results: Patients in CIN group exhibited lower LB%, 25-(OH)D and VitB12 (P < 0.05) and higher Nugent score and H' compared to non-CIN group (P < 0.05). ROC analysis revealed that the AUCs of LB%, Nugent score, H', 25-(OH)D and VitB12 in the diagnosis of CIN were 0.883, 0.907, 0.865, 0.836 and 0.705, respectively (P < 0.05). Pearson analysis found that at admission, LB%, 25-(OH)D and VitB12 in CIN group were negatively correlated with Ki-67 proliferation index and lesion epithelial thickness ratio (P < 0.05), while Nugent score and H' were positively associated with Ki-67 proliferation index and lesion epithelial thickness ratio (P < 0.05). Conclusion: Vaginal microecological imbalance and insufficient serum 25-(OH)D and VitB12 levels are associated with the occurrence and severity of CIN. These indicators may become potential biomarkers for screening and evaluating CIN risk, and provide new ideas for clinical risk assessment.