Abstract: Objective To investigate the clinical application value of BHLHE22 and CDO1 gene methylation detection in cervical exfoliated cells for diognosis of endometrial cancer. Methods Patients who were admitted to Xuzhou Maternal and Child Health Care Hospital between May 2023 and May 2024, and who had suspected endometrial lesions or were diagnosed with endometrial cancer and scheduled for total hysterectomy were recruited. DNA methylation was detected using quantitative methylation-specific PCR (qMSPCR) after sampling with a cervical brush. Results A total of 172 patients were included, where 17 were confirmed to have endometrial cancer, 44 had benign endometrial lesions, 106 had normal endometrium, and 5 had other gynecological cancers. The methylation scores of BHLHE22 and CDO1 genes increased with the progression of endometrial lesions. The sensitivity of BHLHE22 and CDO1 gene methylation detection for endometrial cancer was 94.1%, while the specificity for benign endometrial lesions and normal endometrium was 90.9% and 98.1%, respectively. When all non-endometrial cancer participants were grouped as the "total control" group, the specificity of methylation detection was 94.2%, and the area under the receiver operating characteristic (ROC) curve reached 0.946. One patient whose biopsy pathology indicated endometrial cancer tested negative for methylation due to the removal of the cancerous lesion during hysteroscopic curettage. Conclusions Methylation detection of BHLHE22 and CDO1 genes in cervical exfoliated cells demonstrates excellent sensitivity and specificity in the screening of endometrial cancer. It has the potential to become an objective, reliable, and non-invasive method for endometrial cancer screening.