Development of biosensing platforms plays a key role in research settings for identification of biomarkers and in clinical applications for diagnostics. Biosensors based on nucleic acids have taken many forms, from simple duplex-based constructs to stimuli-responsive nucleic acid nanostructures. In this review, we look at various nucleic acid-based biosensors, the different read-out strategies employed, and their use in chemical and biological sensing. We also look at current developments in DNA nanotechnology-based biosensors and how rational design of such constructs leads to more efficient biosensing platforms.