Background Thrombin is a multifunctional regulatory enzyme of the haemostasis and has both pro- and anticoagulant roles. It has, therefore, been a main target for drug discovery over many decades. Thrombin is a serine protease and possesses two positively charged regions called exosites, through which it is known to bind to many substrates. Dabigatran is a thrombin inhibitor and is widely used as an oral anticoagulant for the antithrombotic treatment of atrial fibrillation and venous thromboembolism. The mechanism by which dabigatran inhibits thrombin is the blockage of the active site, however, its effect on thrombin binding to its substrates has not been studied thoroughly and is thus poorly understood. Material and Methods The effect of dabigatran on thrombin binding to platelets was evaluated by flow cytometry using fluorescently labelled thrombin and washed platelets. Further, to confirm the results we utilized modern techniques for biomolecular binding studies, microscale thermophoresis (MST) and surface plasmon resonance (SPR), which validated the results. Results Dabigatran inhibited thrombin binding to platelets as analysed by flow cytometry. The inhibition was dose dependent with IC50 of 118 nM which was slightly lower than for inhibition of platelet activation and is close to the clinically relevant plasma concentration of dabigatran. MST and SPR also confirmed inhibitory effect of dabigatran on thrombin binding to platelets. Conclusion Apart from blocking the active site, dabigatran also inhibits thrombin binding to platelets. Since thrombin has numerous functions beyond the cardiovascular system, this finding may have important implications.