Precise revealing and early detection of 3-Nitro-L-Tyrosine (3-NLT), a biomarker of oxidative stress in biological media is critical for the early treatment of cancer tumorigenic cells and immunologic disorders. In this study, zinc tungstate (ZnWO4) was incorporated with functionalized carbon nanofibers (f-CNF) to form a ZnWO4/f-CNF composite. The composite improves detection of 3-NLT by increasing the electrical conductivity, electrocatalytic activity, and rapid electron transfer kinetics. Various physical characterization techniques were employed to confirm the ZnWO4/f-CNF composite. Electrochemical impedance spectroscopy, cyclic voltammetry, and differential pulse voltammetry were utilized to detect 3-NLT after modifying ZnWO4/f-CNF on glassy carbon electrode (GCE). The ZnWO4/f-CNF/GCE achieved an elevated electrochemically active surface area (0.08 cm2), a linear range of 1.0-117.0 µM, and a low detection limit of 0.07 µM. Finally, the ZnWO4/f-CNF/GCE was tested with bovine serum albumin and tap water in the real sample investigation.