Rapid and sensitive assay of thrombin and its inhibition in a high-throughput manner is of great significance in the diagnostic and pharmaceutical fields. In this article, we developed a novel biosensor for the detection of thrombin and its inhibition based on the aggregation behavior of the unmodified CdTe QDs. A cationic substrate peptide of thrombin (GGLVPRGSCC-NH2, S-peptide) can attach to the surface of CdTe QDs, partly balance their surface negative charge, and induce the aggregation of QDs, which results in the fluorescence quenching of QDs. After hydrolysis of S-peptide by thrombin, two kinds of shorter peptides (P1-peptide, GGLVPR, and P2-peptide, GSCC) are produced. The uncharged P2-peptide rather than the cationic P1-peptide would bind to QDs. Hence, the CdTe QDs were kept stable in the solution with the fluorescence being maintained. The change of fluorescence intensity would sensitively respond to thrombin activity and its inhibition. Fluorescence spectroscopy, transmission electron microscopy and dynamic light scattering were performed to discuss the quenching mechanism. Under optimized conditions, this method enables measurement of thrombin in the range of 10-100 μU/mL with the detection limit of 1.5 μU/mL. Not only in buffer, but also in blood serum, such sensor exhibited extraordinarily high sensitivity and excellent specificity. In addition, the typical inhibitor of thrombin, hirudin, was also successfully assayed by this method (from 2 μU/mL to 30 μU/mL with the LOD of 0.21 μU/mL). Furthermore, the present approach could also be potentially extended to other proteases and their inhibitors detection with unmodified CdTe QDs.