The ability to accurately detect oral cancer overexpressed 1 (ORAOV 1) offers significant potential for the noninvasive and rapid diagnosis of oral squamous cell carcinoma (OSCC). Present here is an amplified and self-calibrated electrochemical (EC) biosensor for ORAOV 1 detection. This cutting-edge EC biosensor adopts a fuel-driven DNA molecular machine to induce target recycling, thereby amplifying the signals. Meanwhile, by integrating UiO-66 with graphene oxide (GO), the resulting GO@UiO-66 nanomaterials can effectively load substantial amounts of methylene blue (MB), thereby enhancing the EC performance and dramatically improving detection sensitivity. Furthermore, a ratiometric output mode is being developed by using highly conductive GO@UiO-66/MB and silver nanoparticles (Ag NPs) as electroactive tags. By leveraging this ratiometric strategy, which incorporates a built-in self-calibration factor that effectively eliminates systematic errors, the reproducibility, stability, and accuracy of the biosensor can be significantly enhanced. Experimental results demonstrate that this newly developed EC biosensor exhibits ultrahigh sensitivity, with a detection limit as low as 0.28 aM, and a broad linear range from 0.01 fM to 1 pM. Notably, it offers greater reproducibility, stability, and anti-interference capability compared to single-signal output modes. Meanwhile, the detection of ORAOV 1 in human saliva samples has been exemplified with a satisfactory recovery rate and anti-interference capability. More importantly, it can discriminate oral cancer patients from clinical samples with high accuracy (AUC = 1). This approach provides new insights into the noninvasive and early diagnosis of OSCC in clinical applications.