Herein, by directly using Watson-Crick base pairing, a highly ordered and field-free three-dimensional (3D) DNA nanostructure is self-assembled by azobenzene (azo)-functionalized DNA nippers in a few minutes, which was applied as a 3D DNA nanomachine with an improved movement efficiency compared to traditional Au-based 3D nanomachines due to the organized and high local concentration of nippers on homogeneous DNA nanostructure. Once microRNA (miRNA) interacts with the 3D nanomachine, the nippers "open" to hybridize with the miRNA. Impressively, photoisomerization of the azo group induces dehybridization/hybridization of the nippers and miRNA under irradiation at different wavelengths, which easily solves one main technical challenge of DNA nanotechnology and biosensing: reversible locomotion in one step within 10 min. As a proof of concept, the described 3D machine is successfully applied in the rapid single-step detection of a biomarker, which gives impetus to the design of new generations of mechanical devices beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies.