This paper illustrates a new and sensitive electrochemical immunosensor for the analysis of C-reactive protein. Indium Tin Oxide (ITO) disposable sheets were modified by using 3-cyanopropyltrimethoxysilane (CPTMS) self-assembled monolayers (SAMs) for the first time for immobilizing the anti-CRP antibody via covalent interactions without the need for any cross-linking agent. Cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS), as well as square wave voltammetry (SWV) methods were applied to characterize immobilization steps of anti-CRP and to determine the CRP concentration. The optimization of the fabricated parameters and the analytical performance of the biosensor were widely evaluated. Charge transfer resistance changes were highly linear and sensitive with CRP concentration of 3.25–208fgmL−1 range and associated with a limit of detection of 0.455fgmL−1. This impedimetric biosensing system have excellent repeatability, reproducibility and reusability. Moreover, the binding characterization of CRP to anti-CRP was monitored by a single frequency impedance technique. The amount of CRP in human serum samples were analyzed by fabricated biosensor to determine the feasibility of the biosensing system in medical purposes. We suggest that CPTMS, a new silanization agent, is ideal in biosensor applications.