Inspired by molecular machines in nature, artificial nanodevices have been designed to realize various biomedical functions. Self-assembled deoxyribonucleic acid (DNA) nanostructures that feature designed geometries, excellent spatial accuracy, nanoscale addressability and marked biocompatibility provide an attractive candidate for constructing dynamic nanodevices with biomarker-targeting and stimuli-responsiveness for biomedical applications. Here, a summary of typical construction strategies of DNA nanodevices and their operating mechanisms are presented. We also introduced recent advances in employing DNA nanodevices as platforms for biosensing and intelligent drug delivery. Finally, the broad prospects and main challenges of the DNA nanodevices in biomedical applications are discussed.