Nucleic acid-based biosensors for the detection of specific proteins combine the typical programmability of synthetic DNA systems with artificially controlled DNA-protein communication. The high-affinity interaction between a target protein and a specific ligand, such as an aptamer sequence, or a double stranded DNA domain, or a small peptide, is paired with a nature-mimicking molecular mechanism allowing for probing, processing, and translating protein binding activity into a measurable signal. In this Review, two main strategies developed in the context of protein-responsive nucleic acid-based biosensors are discussed. One is the design of proximity-based assays harnessing the spatial colocalization of functional probes within the volume of a multivalent protein. The other is the engineering of dynamic DNA structures that undergo a controlled conformational or structural change upon protein binding. Examples of applications from optical and electrochemical detection of antibodies in biofluids to fluorescence imaging of transcription factors in living cells are reported, and suggestions along with possible future directions in the field are discussed.