Thrombin is an important enzyme that plays a pivotal role in the blood clotting pathways. An imbalance in the activity of this enzyme is clinically known to be associated with various diseases, such as thrombosis, inflammation, atherosclerosis, and haemophilia, suggesting the need to devise sensors for Thrombin detection. However, the majority of the fluorescence-based Thrombin assays rely on fluorescence labelling assays or Thrombin specific recognition biomolecules such as aptamers or antibody which requires sophisticated techniques and makes it very expensive. Herein, we report a simple, selective, sensitive and label-free fluorescence detection scheme for Thrombin which is based on the interaction between Thrombin and a fluorescent complex of Heparin with a molecular rotor dye, Thioflavin-T. The detection scheme exploits selective interaction between cationic Thrombin and anionic Heparin to modulate the monomer-aggregate equilibrium of the Thioflavin-T-Heparin system. Importantly, the present system offers a ratiometric response that has the ability for robust quantification of Thrombin concentration even in complex medium. The involvement of all commercially available components is a crucial advantage of this detection scheme. Further, the detection scheme also shows reasonable response in diluted serum matrix.Copyright © 2018. Published by Elsevier B.V.