A simple and ultrasensitive label-free electrochemical impedimetric aptasensor for thrombin based on the cascaded signal amplification was reported. The sandwich system of aptamer/thrombin/aptamer-functionalized Au nanoparticles (Apt-AuNPs) was fabricated as the sensing platform. The change of the interfacial feature of the electrode was characterized by electrochemical impedance analysis with the redox probe [Fe(CN)(6)](3-/4-). For improving detection sensitivity, the three-level cascaded impedimetric signal amplification was developed: (1) Apt-AuNPs as the first-level signal enhancer; (2) the steric-hindrance between the enlarged Apt-AuNPs as the second-level signal amplification; (3) the electrostatic-repulsion between sodium dodecylsulfate (SDS) stabilized Apt-AuNPs and the redox probe [Fe(CN)(6)](3-/4-) as the third-level signal amplification. Enlargement of Apt-AuNPs integrated with negatively charged surfactant (SDS) capping could not only improve the detection sensitivity of the impedimetric aptasensor for thrombin but also present a simple and general signal-amplification model for impedimetric sensor. The aptasensor based on the enlargement of negatively charged Apt-AuNPs showed an increased response of the electron-transfer resistance to the increase of thrombin concentration through a wide detection range from 100 fM to 100 nM. The linear detection range was 0.05-35 nM, and thrombin was easily detectable to a concentration of 100 fM. The aptasensor also has good selectivity and reproducibility.