The aim of this research was to design an electrochemical immunosensor for determination of tumour necrosis factor receptor-associated protein-1(TRAP1) antigen, a heat shock protein linked to tumour necrosis factor. The indium-tin oxide covered polyethylene terephthalate (ITO-PET) electrode surface was cleaned and was prepared for the introduction of hydroxyl groups on its surface by using NH4OH/H2O2/H2O. As a silanization agent for covalent attachment of anti-TRAP1 on the surface of the ITO working electrode, 3-glycidoxypropyltrimethoxysilane (3-GOPS) was used. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the immobilization steps. A variety of parameters, 3-GOPS and anti-TRAP1 concentrations, and anti-TRAP1 and TRAP1 incubation durations were optimized. After determining the optimum conditions, characterization studies such as repeatability, reproducibility, regeneration, square wave voltammetry, and single frequency impedance were performed. The electrochemical immunosensor has presented an extremely wide determination range for TRAP1 from 0.1 pg/mL to 100 pg/mL.