Due to the incorporation of gold nanoparticles (AuNPs), previously reported AuNP-based FRET nanoflares still have some problems, such as non-negligible cytotoxicity and a time-consuming preparation procedure. In this communication, a novel AuNP-free FRET nanoflare for intracellular ATP imaging is developed based on a DNA nanostructure, which is self-assembled through cyclic U-type hybridization only involving a certain number of DNA strands.