A new flexible biosensor based on conjugated polymer functionalized indium tin oxide (ITO) sheet was fabricated for Receptor for Activated C Kinase 1 (RACK 1) determination. Poly(3-thiophene acetic acid) (P(Thi-Ac)) was used as an immobilization matrix for construction of RACK 1 immunosensor. This polymer had a great number of carboxyl groups on its end site and these carboxyl ends provided anchoring points to the anti-RACK 1 antibodies. Anti-RACK 1 antibodies were covalently attached on the ITO electrode and recognized the RACK 1 antigens. Electrochemical characterizations were made by employing electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. Additionally, single frequency impedance method (SFI) was applied to follow the specific biointeraction between antibody and antigen. As a result of specific biointeraction, the designed immunosensor exhibited a wide linear detection range between 0.01 pg/mL and 2 pg/mL RACK 1 with a detection limit of 3.1 fg/mL. Scanning electron microscopy and atomic force microscopy analyses were employed for electrode surface morphology investigation. The designed RACK 1 biosensor had good repeatability (5.73 %, RSD), excellent reproducibility (2.5 %, RSD), long storage-stability and reusable property. In addition, the fabricated RACK 1 biosensor was applied to determine RACK 1 concentration in human serums and the recovery was ranging from 98.79%-100.22%. This work illustrated a new tool to construct a sensitive and low-cost disposable biosensor for applications in clinical monitoring. (C) 2020 Elsevier B.V. All rights reserved.