A copper nanoclusters (CuNCs)-based fluorescence biosensing platform was developed for label-free DNA detection through a cascade isothermal exponential amplification reaction (EXPAR). It is based on target-triggered isothermal dual-cycling and a cascade EXPAR, which generates a large amount of long double-stranded DNA (dsDNA). The resultant long dsDNA can act as an efficient template for fluorescent CuNC synthesis, and the produced CuNC is an excellent fluorescence indicator to quantify target DNA concentration. This assay allows the detection of the model DNA with a detection limit of 7 pM. This label-free assay occurs in a homogenous solution, and the whole assay process, including hybridization, amplification and detection, can be completed in a single-tube. This method is simple, low-cost and convenient to operate, and may offer potential applications in related biomedical research.