Interactions between biomolecules are an important feature of biological systems and understanding these interactions is a key goal in biochemical studies. Using conventional techniques, such as surface plasmon resonance and isothermal titration calorimetry, the determination of the binding constants requires a significant amount of time and resources to produce and purify sufficient quantities of biomolecules in order to measure the affinity of biological interactions. Using DNA hybridization, we have demonstrated a new technique based on the use of nanotethers and time-resolved Forster resonance energy transfer (FRET) that significantly reduces the amount of material required to carry out quantitative binding assays. Test biomolecules were colocalized and attached to a surface using DNA tethers constructed from overlapping oligonucleotides. The length of the tethers defines the concentration of the tethered biomolecule. Effective end concentrations ranging from 56 nM to 3.8 μM were demonstrated. The use of variable length tethers may have wider applications in the quantitative measurement of affinity binding parameters.