This paper presents a high specific, sensitive electrochemical biosensor for recognition of protein such as thrombin based on aptamers and nano particles. Two different aptamers were chosen to construct a sandwich manner for detecting thrombin. Aptamer Ⅰ was immobilized on nano magnetic particle for capturing thrombin, and aptamer Ⅱ labled with nano gold was used for detection. The electrical current generated from gold after the formation of the complex of magnetic particle, thrombin and nano gold,and then an electrochemical cell designed by ourselves was used for separating, gathering, and electrochemical detecting. Through magnetic separation, high specific and sensitive detection of the target protein, thrombin, was achieved. Linear response was observed over the range 5.6×10-12-1.12×10-9mol/L, with a detection limit of 1.42×10-12 mol/L. The presence of other protein as BSA did not affect the detection, which indicates that high selective recognition of thrombin can be achieved in complex biological samples such as human plasma.