Progress in understanding and treatment of thrombotic diseases requires new effective methods for the easy, rapid, and reversible control of coagulation processes. In this framework, the use of aptamers, and particularly of the thrombin binding aptamer (TBA), has aroused strong interest, due to its enormous therapeutic potential, associated with a large number of possible applications in biotechnological and bioanalytical fields. Here, we describe a new TBA analogue (named tris-mTBA), carrying three different pendant groups: a dansyl residue at the 3 '- and a beta-cyclodextrin moiety at the 5 '-end-providing a host-guest system which exhibits a marked fluorescence enhancement upon TBA G-quadruplex folding-and a biotin tag, allowing the attachment of the aptamer onto biocompatible streptavidin-coated silica nanoparticles (NPs) of 50 nm hydrodynamic diameter (Sicastar). The use of nanoparticles for the in vivo delivery of TBA, expected to induce per se increased nuclease resistance and improved pharmacokinetic properties of this oligonucleotide, offers as an additional advantage the possibility to exploit multivalency effects, due to the presence of multiple copies of TBA on a single scaffold. In addition, the selected fluorescent system allows monitoring both the presence of TBA on the functionalized NPs and its correct folding upon immobilization, also conferring enhanced enzymatic resistance and bioactivity. The anticoagulant activity of the new tris-mTBA, free or conjugated to Sicastar NPs, was evaluated by dynamic light scattering experiments. Highly effective and reversible inhibition of thrombin activity toward fibrinogen was found for the free tris-mTBA and especially for the tris-mTBA-conjugated NPs, demonstrating great potential for the biomedical control of blood clotting.