Binary light-up aptamers are intriguing and emerging tools with potential in different fields. Herein, we demonstrate the versatility of a split Broccoli aptamer system able to turn on the fluorescence signal only in the presence of a complementary sequence. First, an RNA three-way junction harbouring the split system is assembled in anE. coli-based cell-free TX-TL system where the folding of the functional aptamer is demonstrated. Then, the same strategy is introduced into a ‘bio-orthogonal’ hybrid RNA/DNA rectangle origami characterized by atomic force microscopy: the activation of the split system through the origami self-assembly is demonstrated. Finally, our system is successfully used to detect the femtomoles of aCampylobacterspp. DNA target sequence. Potential applications of our system include the real-time monitoring of the self-assembly of nucleic-acid-based devicesin vivoand of the intracellular delivery of therapeutic nanostructures, as well as thein vitroandin vivodetection of different DNA/RNA targets.