A novel ratiometric electrochemical aptasensor for sensitive and selective analysis of thrombin has been developed based on aptamer target aptamer sandwich structure. In the absence of thrombin, the methylene blue (MB) tagged thrombin aptamer (MB-TBA) was introduced on the electrode through the hybridization with the immobilized DNA sequences, thereby observing an oxidation signal of MB due to the flexible, single-stranded element of 5' end of MB-TBA. After the addition of thrombin, the specific thrombin aptamer interaction drove the conformational change of MB-TBA (from the unfolded conformation to the G-quadruplex), resulting in the MB tag far from the electrode surface. The second thrombin aptamer with ferrocene (Fc) tagged on both chain ends (Fc-TBA-Fc) interacted with thrombin to form another G-quadruplex conformation. Such sandwich structure and the G-quadruplex conformation acted cooperatively to allow the two Fc tags to be close to the electrode surface. These conformational changes resulted in a decrease of the oxidation peak current of MB (I-MB) and an increase of that of Fc (I-Fc), and the logarithmic value of I-Fc/I-MB was linear with the logarithm of thrombin concentration. The developed aptasensor shows good response toward the target with a wide linear range from 1 nM to 600 nM and a low detection limit of 170 pM. (C) 2014 Elsevier B.V. All rights reserved.