This investigation displays a novel, practical indium tin oxide- polyethylene terephthalate (ITO-PET) based electrochemical biosensor for the Tumor Necrosis Factor-alpha (TNFα) biomarker determination. The ITO-PET electrode is a very advantageous preferred semiconductive electrode material. It has a lot of great features such as easy to prepare, cheapness, flexibility, stability. It also allows determining an analyte at very low concentrations and provides a great wide concentration range for analyte analysis. Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV) were used for the evaluation of biosensors, including the immobilization procedure, the investigation of the optimum conditions, and the characterization of biosensors. The immunosensor's electrode surface morphology during the immobilization process was observed using a Scanning Electron Microscope (SEM). In addition, Impedance measurement at a single frequency was used to characterize anti-TNFα and TNFα interactions (SFI). The clinical effectiveness of the developed biosensor was investigated by testing it with real human serum samples. Moreover, the fabricated immunosensor presents long shelf life, analysis of the antigen concentrations at picogram level (0.02 pg mL-1 -2.56 pg mL-1), reproducibility, reusability (11 times) and high sensitivity.