In this study, we constructed a new and sensitive ITO based electrochemical immunosensor for detection of interleukin 1 beta(IL-1 beta), a cancer biomarker found in serum and saliva. 6-phosphonohexanoic acid (PHA) was used as a biomolecule immobilization matrix for the first time. Anti-IL-1 beta antibody was utilized as a biorecognition molecule that immobilized onto carboxyl groups of 6-phosphohexanoic acid (PHA) via amide bond. Selective interaction between anti-IL-1 beta antibodies and IL-1 beta antigens was investigated by Electrochemical Impedance Spectroscopy (EIS), Cyclic Voltammetry (CV) and Single Frequency Impedance (SFI) methods. The surface characterization of the immunosensor was performed by fourier transform infrared spectroscopy (FTIR), raman spectroscopy, scanning electron microscopy (SEM), SEM-energy dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM) in order to illustrate individual steps of biosensor construction. Under the optimized experimental conditions, the change in impedance was proportional to IL-1 beta concentrations in the range of 0.025-3 pg/mL (R-2 = 0.99) with detection limit of 7.5 fg/mL. The reproducibility, repeatability, stability, and specificity of the developed immunosensor were analyzed. In addition, the developed immunosensor was successfully utilized for the determination of IL-1 beta in serum and saliva samples by using the standard addition method with recoveries of 96.7-105.4%. This immunosensor was applicable for the requirements of routine analysis with respect to performance, functionality and cost. (C) 2018 Elsevier B.V. All rights reserved.