In this study, we developed a simple, sensitive, low-cost and label-free method to detect rutin by using double-stranded DNA-templated copper nanoclusters (dsDNA-CuNCs) as a fluorescent probe. The dsDNA-template can facilitate the formation of highly fluorescent CuNCs with a maximum emission wavelength of 575 nm. In the dsDNA-CuNC fluorescent probe, rutin was intercalated into the structure of dsDNA via hydrogen bonding and electrostatic interactions. The fluorescence of dsDNA-CuNCs was quenched considerably due to the electron transfer from CuNCs to rutin. This decrease in the fluorescence intensity of dsDNA-CuNCs can be used to monitor the concentration of rutin from 0.6 to 400 μM with a detection limit of 0.12 μM (S/N = 3). Compared with the previous reports, this method does not require any complex DNA sequence design, fluorescent dye label and sophisticated experimental techniques. Moreover, the proposed sensing system could be applicable for the detection of rutin in pharmaceutical samples.