To detect the target molecules, aptamers are currently focused on and the use of aptamers for biosensing is particularly interesting, as aptamers could substitute antibodies in bioanalytical sensing. So this paper describes the novel electrochemical system for protein in sandwich manner by using the aptamers and the scanning electrochemical microscope (SECM). For protein detection, sandwich system is ideal since labeling of the target protein is not necessary. To develop the electrochemical protein sensor system, thrombin was chosen as a target protein since many aptamers for it were already reported and two different aptamers, which recognize different positions of thrombin, were chosen to construct sandwich type sensing system. In order to obtain the electrochemical signal, the glucose oxidase (GOD) used for labeling the detection aptamers since it has large amount of stability in aqueous solution. One aptamer was immobilized onto the gold electrode and the other aptamer for detection was labeled with GOD for generation of the electric signal. Thrombin was detected in sandwich manner with aptamer immobilized onto the gold electrode and the GOD labeled aptamer. The enzymatic signal, generated from glucose addition after the formation of the complex of thrombin, was measured. The generation-collection mode of SECM was used for amperometric H(2)O(2) detection.