Thrombin plays a critical role in hemostasis and hemolysis. It is of high importance to develop a system toward thrombin detection with high sensitivity and high selectivity for both research and clinical diagnosis applications. In this paper, we developed a thrombin detection assay by taking advantage of the novel signal amplified strategy based on the chiral supramolecular assembly in physiological K background. This assay could detect thrombin as low concentration as about 2 pM and provided a highly specific selectivity among several common interferences. Furthermore, the assay can discriminate thrombin from other nonspecific analogous proteins with high selectivity and can be used to detect thrombin in diluted real human serum samples, which suggested its great potential for rapid detection of thrombin in the clinic.