We demonstrate the use of a novel electrochemical sensing platform based on aptamer conjugated Mo6S9−xIx nanowires (MoSI NWs) for the highly sensitive detection of the blood clotting enzyme thrombin.MoSI NWs nanowires were self-assembled on a gold electrode to which thrombin binding aptamers were covalently attached. Themodiﬁcation and immobilization steps of the electrodeswere characterised by cyclic voltammetry along with high-resolution transmission electron microscopy and X-ray photoelectron spectroscopy. The platform is based on the creation of a self-assembled MoSI MWlayer via thesulfur–gold afﬁnity followed by the creation of MoSI-thiolated aptamer conjugates via the sulfur–sulfur afﬁnity. Using this system, sensitive quantitative detection of thrombin is realized by monitoring differences of differential pulse voltammetric responses of electrostatically trapped [Ru(NH3)6]3+ cations to the aptamer before and after thrombin binding. The sensitivity limit for the detection of thrombin is 10 pM. This value is 10-fold better than all currently reported one step label free electrochemical strategies. Given the direct label free nature of the approach and the simplicity of the electronic detection, theaptamer conjugated MoSI NWs biosensor appears well suited for implementation in portable point of care microdevices directed at the rapid and sensitive detection of proteins and pathogens.