In this study, a sensitive immunosensor was developed for the first time for p21-activated kinase 2 (PAK 2) detection. In the design of the immunosensor, 3-glycidoxypropyltrimethoxysilane (GPTMS) was utilized as an ITO electrode modification material for anti-PAK 2 antibody immobilization. This molecule had epoxy group, which was reactive to amino groups of antibodies. Anti-PAK 2 antibodies were also used as biomolecules for sensitive interaction for PAK 2 antigen. In the presence of PAK 2 antigens, high impedance signal was observed when the process followed by using Electrochemical Impedance Spectroscopy technique (EIS). Apart from EIS technique, Cyclic Voltammetry (CV), Square Wave Voltammetry (SWV) and Single Frequency Impedance (SFI) techniques were utilized. Microscopic surface characterizations of immunosensor fabrication steps were performed by using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). The immunosensor exhibited good sensitivity and selectivity for PAK 2 antigen detection. A linear calibration curve between EIS response and PAK 2 concentration was obtained in the range of 0.005-0.075 pg/mL with the detection limit of 1.5 fg/mL. It had good repeatability, excellent reproducibility and high stability. Additionally, this immunosensor can be reused by simple application protocol. Furthermore, it had good recovery for PAK 2 antigen detection in human serum samples. The good recovery illustrated that the developed immunosensor was a promising tool for PAK 2 detection in practical applications. (C) 2019 Elsevier B.V. All rights reserved.