A novel impedimetric aptasensor using a mixed self-assembled monolayer composed of thiol-modified thrombin binding aptamer and 2-mercaptoethanol on a gold electrode is reported. The changes of interfacial features of the electrode were probed in the presence of the reversible redox couple, Fe(CN)6(3-/4-), using impedance measurements. The electrode surface was partially blocked due to the self-assembly of aptamer or the formation of the aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by electrochemical impedance spectroscopy or cyclic voltammetry. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution, and the immobilized aptamer subsequently was used for repeated detection of thrombin. The aptamer-functionalized electrode showed a linear response of the charge-transfer resistance to the increase of thrombin concentration in the range of 5.0-35.0 nM and the thrombin was easily detectable to a concentration of 2.0 nM.