DNA nanotechnology has progressed from proof-of-concept demonstrations of structural design towards application-oriented research. As a natural material with excellent self-assembling properties, DNA is an indomitable choice for various biological applications, including biosensing, cell modulation, bioimaging and drug delivery. However, a major impediment to the use of DNA nanostructures in biological applications is their susceptibility to attack by nucleases present in the physiological environment. Although several DNA nanostructures show enhanced resistance to nuclease attack compared with duplexes and plasmid DNA, this may be inadequate for practical application. Recently, several strategies have been developed to increase the nuclease resistance of DNA nanostructures while retaining their functions, and the stability of various DNA nanostructures has been studied in biological fluids, such as serum, urine and cell lysates. This Review discusses the approaches used to modulate nuclease resistance in DNA nanostructures and provides an overview of the techniques employed to evaluate resistance to degradation and quantify stability.