A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin. The signal enhancement was achieved by using gold nanoparticles (GNPs), which was electrodeposited onto a glassy carbon electrode (GCE), as a platform for the immobilization of the thiolated aptamer. In the measurement of thrombin, the change in interfacial electron transfer resistance of the biosensor using a redox couple of [Fe(CN)(6)](3-/4-) as the probe was monitored. The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12nM to 30nM. The association and dissociation rate constants of the immobilized aptamer-thrombin complex were 6.7x10(3)M(-1)s(-1) and 1.0x10(-4)s(-1), respectively. The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed. This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein. This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE.