<p>Oral cancer presents substantial challenges to global health due to its elevated mortality rates. Approximately 90% of these malignancies are oral squamous cell carcinoma (OSCC). A significant contributor to the prevalence of oral cancer is the difficulty in detecting cancerous biomarkers, further exacerbated by socioeconomic disadvantages and late-stage diagnoses. Given the critical nature of oral cancer, the early detection of biomarkers is essential for reducing mortality rates. This study investigates the application of microRNA-31 (miRNA-31) as a biomarker for the electrochemical detection of oral cancer, recognizing the considerable potential that microRNAs have demonstrated in cancer screening and diagnosis. The methodology employed includes the use of a glassy carbon electrode modified with graphene and a molecular tethering agent designed to enhance sensitivity and specificity. The biosensor exhibited a limit of detection of 10^− 11 M (70 pg/mL or 6.022 × 10⁶ copies/µL) in buffer and 10^− 10 M (700 pg/mL or 6.022 × 10⁷ copies/µL) in diluted serum for the complementary target miRNA-31 using the Six Sigma method. The efficacy of this biosensor was further validated through specificity studies utilizing a non-complementary miRNA in both buffer and human serum samples. The electrochemical biosensor displayed exceptional performance and high sensitivity in detecting miRNA-31, confirming its role as an innovative sensor for the non-invasive diagnosis of oral cancer. Furthermore, the proposed biosensor demonstrates several advantages over current methodologies, including reduced detection time, and cost-effective reagents.</p> Graphical Abstract <p></p>