We report the quantitative measurement of aptamer-protein interactions using backscattering interferometry (BSI) and show that BSI can determine when distinct binding regions are accessed. As a model system, we utilized two DNA aptamers (Tasset and Bock) that bind to distinct sites of a target protein (human α-thrombin). This is the first time BSI has been used to study a multivalent system in free solution wherein more than one ligand binds to a single target. We measured aptamer equilibrum dissociation constants (K(d)) of 3.84 nM (Tasset-thrombin) and 5.96 nM (Bock-thrombin), in close agreement with the literature. Unexpectedly, we observed allosteric effects such that the binding of the first aptamer resulted in a significant change in the binding affinity of the second aptamer. For example, the K(d) of Bock aptamer binding to preformed Tasset-thrombin complexes was 7-fold lower (indicating higher affinity) compared to binding to thrombin alone. Preliminary modeling efforts suggest evidence for allosteric linkage between the two exosites.