A new, quite sensitive disposable immunosensor, based on the anti-RACK1 antibody, was developed for the determination of Receptor for Activated C Kinase 1 (RACK1) for the first time. Moreover, indium tin oxide (ITO) flexible sheets were modified by using aldehyde ended silane 11-(triethoxysilyl) undecanal (TESU) self-assembled monolayers (SAMs) for immobilizing the anti-RACK1 antibody via covalent bonds. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square wave voltammetry (SWV) methods were used for characterizing the immobilization steps and for determining the RACK1 concentration. To obtain a successful immunosensor, experimental factors were optimized. This quite sensitive biosensor was able to detect concentrations as low as fg mL(-1). The detection principle was based on the change in charge transfer resistance and the Nyquist plot diameter and these changes were monitored by using electrochemical impedance spectroscopy (EIS). This impedimetric immunosensor had perfect repeatability and reproducibility. We proved that the new silane agent, TESU, performs well in biosensor applications. The feasibility of the fabricated immunosensor was tested by detecting RACK1 in artificial and real human serum samples.