Continuous glucose monitoring is valuable for people with diabetes but faces limitations due to enzyme–electrode interactions and biofouling from biological samples that reduce sensor sensitivity and the monitoring performance. We created an enzyme-based electrochemical system with a unique nanocomposite coating that incorporates the redox molecule, aminoferrocene (NH2-Fc). This coating enhances stability via electroactivity and reduces nonspecific binding, as demonstrated through cyclic voltammetry. Our approach enables real-time glucose detection via chronoamperometry with a calculated linear range of 0.5 to 20 mM and a 1 mM detection limit. Validated with plasma and saliva, this platform shows promise for robust metabolite detection in clinical and research contexts. This versatile platform can be applied to accurately monitor a wide range of metabolites in various biological matrices, improving patient outcomes.