DNA–protein interactions may be observed on single molecules with a variety of techniques. However, quantifying the binding affinity is difficult and often requires many (∼100) individual events to characterize the interaction. We use a single λ DNA molecule that provides a lattice of binding sites for proteins. Extending and relaxing the tethered molecule reversibly melts DNA, allowing it to be converted between double-stranded (ds) and single-stranded (ss) forms. By monitoring changes in the properties of the DNA as a function of added protein concentration and fitting to binding models, the DNA–protein interaction may be characterized and quantified. As an example, the high mobility group protein HMGB1(box A  +  B) is observed to stabilize dsDNA. Measuring the strength of this effect allows us to determine the equilibrium association constant for HMGB1(box A  +  B) binding to dsDNA.