Thrombin-binding aptamer-conjugated gold nanoparticles (TBA-Au NPs) for highly effective control of thrombin activity towards fibrinogen are demonstrated. While a 29-base long oligonucleotide (TBA29) has known no enzymatic inhibitory functions for thrombin-mediated coagulation, the ultrahigh anticoagulant potency of TBA29-Au NPs can be demonstrated via the steric blocking effect, at two orders of magnitude higher than that of free TBA29. The surface aptamer density on the Au NPs is important in determining their enzymatic inhibition of thrombin and their stability in the presence of nuclease. The practicality of 100TBA29-Au NPs (100 TBA29 molecules per Au NP) for controlling thrombin-mediated coagulation in plasma is found, and the 100TBA29-Au NPs has an ultra binding affinity towards thrombin (Kd = 2.7 × 10−11M) due to their high ligand density. The anticoagulant activity of TBA29-Au NPs is found to be suppressed by TBA29 complementary sequence (cTBA29) modified Au NPs (cTBA29-Au NPs), with a suppression rate 4.6-fold higher than that of cTBA29. The easily prepared and low-cost TBA29-Au NPs and cTBA29-Au NPs show their potential in biomedical applications for treating various diseases related to blood clotting disorders. In principle, this study opens the possibility of regulation of molecule binding, protein recognizing, and enzyme activity by using aptamer-functionalized nanomaterials.