A label-free electrochemical impedance spectroscopy (EIS) biosensor for highly sensitive detection of cardiac troponin I (TnI) as a model target was developed by employing gold nanoparticle (GNPs) modified glassy carbon electrode (GCE) as a base electrode. A special peptide (CFYSHSFHENWPS) was used as molecular recognition probe. GNPs were firstly electrochemically deposited onto the surface of GCE and polyethylene glycol was then used to block any remaining sites of the surface of GCE. The biosensor was fabricated by thiol self-assembling the peptide probe onto the surface of gold nanoparticles and finally blocking any remaining sites of the surface of GNPs using 6-mercapto-1-hexanol. A suitable equivalent electrical circuit consisting of parallel double Randles circuit with phase angle elements greatly fits the experiment electrochemical cell. Upon the binding of TnI, the charge transfer resistance (R-ct) of the biosensor was logarithmically direct proportional to the concentration of TnI in the range from 15.5 pg/mL to 1.55 ng/mL with a detection limit of 3.4 pg/mL. It was found that GNPs modified GCE as a platform for the immobilization of the peptide can greatly reduce the background EIS signal and enhance the EIS response to the target. This biosensor has been used for assay of real serum sample with satisfactory results. This work demonstrates that GNPs modified GCE for the immobilization of molecular recognition element is promising platform for the development of label-free electrochemical biosensors for the determination of special proteins. (C) 2016 Elsevier B.V. All rights reserved.