Thrombin binding aptamers HD1 and HD22 are the most studied aptamers, both for therapeutic and sensing purposes. Yet there is still no commercialized aptamer-based sensor device for thrombin detection, suggesting that the binding modes of these aptamers remain to be precisely described. Here we investigate thrombin-aptamer interactions with molecular dynamics simulations, and show that the different solved structures of HD1-thrombin complex are energetically similar and consequently possibly co-existing. Conversely HD22 folding is much more stable and its binding energy with thrombin is significantly largerthan that of HD1 complexes. These results are confronted to experiments, which consist in monitoring aggregation of aptamer-functionalized gold nanoparticles triggered by thrombin. HD1 alone, but not HD22, can trigger aggregation, meaning that this aptamer has multiple sites of interactions with thrombin. Furthermore, pre-incubation of HD22 with thrombin impedes HD1 aggregation, suggesting that HD1 and HD22 have competing affinities for the same binding site. Altogether this study shows that thecharacterization of aptamer-thrombin interactionsby structural and kinetic experiments joined to simulations is necessary for the development of biosensors. Aptamers are single-stranded nucleic acids able to bind to a variety of targets with high speci-ficity and affinity 1-3. Their selection is achieved through the SELEX protocol, which consists in consecutivecycles of selection and amplification, starting from a large library of oligonuc-leotides (10 13 –10 18 sequences) 4. Due to their attractive properties in term of ease of synthesis, low-cost, chemical stability, and high affinity and specificity for a broad range of targets, ap-tamers have attracted much attention, particularly as potential therapeutic inhibitors and sensing elements for a future generation of biosensors 5. Thrombin binding aptamers (TBA) HD1 and HD22 have been the most studied aptamers for biosensing, representing ~20% of the 5000 papers published about the use of aptamers for analytical technologies 6 .Surprisingly there is little consensus on their binding properties in the literature. A rapid survey of Surface Plasmon Resonance (SPR) data shows that the reaction constant of HD1 and HD22 spans ~2