AbstractIn order to improve the survival rate of oral squamous cell carcinoma (OSCC) patients, a reliable diagnostic method for early OSCC detection is required that is minimally invasive, less burdensome to the patient, and has high sensitivity and specificity. Therefore, we performed the detection of abnormal methylation at three locations in the hTERT promoter region of oral exfoliated cells by employing the ferrocenylnaphthalene diimide (FND)‐based electrochemical hybridization assay (FND‐EHA) using three types of DNA probe‐immobilized electrodes. We also performed liquid cytology using oral exfoliated cells and compared these obtained data to evaluate whether FND‐ EHA can be used as an OSCC screening system. The results showed a good correlation between this method and conventional OSCC screening, and cytology. In addition, FND‐EHA was also able to determine samples that had been ambiguously determined by liquid cytology. This indicates that FND‐EHA may be useful as an OSCC screening system.