An accurate and genome-wide characterization of protein–DNA interactions such as transcription factor binding is of utmost importance for modern biology. Powerful screening methods emerged. But the vast majority of these techniques depend on special labels or markers against the ligand of interest and moreover most of them are not suitable for detecting low-affinity binders. In this article a molecular force assay is described based on measuring comparative unbinding forces of biomolecules for the detection of protein–DNA interactions. The measurement of binding or unbinding forces has several unique advantages in biological applications since the interaction between certain molecules and not the mere presence of one of them is detected. No label or marker against the protein is needed and only specifically bound ligands are detected. In addition the force-based assay permits the detection of ligands over a broad range of affinities in a crowded and opaque ambient environment. We demonstrate that the molecular force assay allows highly sensitive and fast detection of protein–DNA interactions. As a proof of principle, binding of the protein EcoRI to its DNA recognition sequence is measured and the corresponding dissociation constant in the sub-nanomolar range is determined. Furthermore, we introduce a new, simplified setup employing FRET pairs on the molecular level and standard epi-fluorescence for readout. Due to these advancements we can now demonstrate that a feature size of a few microns is sufficient for the measurement process. This will open a new paradigm in high-throughput screening with all the advantages of force-based ligand detection.