Diabetes is widely considered as a silent killer which affects the internal organs and ultimately has drastic impacts on our day-to-day activities. One of the fatal outcomes of diabetes is diabetic foot ulcer (DFU); which, when becomes chronic, may lead to amputation. The incorporation of nanotechnology in developing bio-sensors enables the detection of desired biomarkers, which in our study are glucose and L-tyrosine; which gets elevated in patients suffering from diabetes and DFUs, respectively. Herein, we report the development of an enzymatic impedimetric sensor for the multi-detection of these biomarkers using an electrochemical paper-based analytical device (µ-EPADs). The structure consists of two working electrodes and a counter electrode. One working electrode is modified with α-MnO2-GQD/tyrosinase hybrid to aid L-tyrosine detection, while the other electrode is coated with α-MnO2-GQD/glucose oxidase hybrid for glucose monitoring. Electrochemical impedance spectroscopy has been employed for the quantification of glucose and L-tyrosine, within a concentration range of 50–800 mg/dL and 1–500 µmol/L, respectively, using a sample volume of approximately 200 µL. The impedance response exhibited a linear relationship over the analyte concentration range with detection limits of ~58 mg/dL and ~0.3 µmol/L for glucose and tyrosine respectively, with shelf life ~1 month. The sensing strategy was also translated to Arduino-based device applications by interfacing the µ-EPADs with miniaturized electronics.