Many proteins form dynamic complexes with DNA, RNA, and other proteins, which often involves protein conformational changes that are key to function. Yet, methods to probe these critical dynamics are scarce. Here we combine optical tweezers with fluorescence imaging to simultaneously monitor the conformation of individual proteins and their binding to partner proteins. Central is a protein–DNA coupling strategy, which uses exonuclease digestion and partial re-synthesis to generate DNA overhangs of different lengths, and ligation to oligo-labeled proteins. It provides up to 40 times higher coupling yields than existing protocols and enables new fluorescence-tweezers assays, which require particularly long and strong DNA handles. We demonstrate the approach by detecting the emission of a tethered fluorescent protein and of a molecular chaperone (trigger factor) complexed with its client. We conjecture that our strategy will be an important tool to study conformational dynamics within larger biomolecular complexes.@en