By taking advantage of the large signal amplification through efficient fluorescence resonance energy transfer (FRET) from the conjugated polymer to the intercalating dye mediated by DNA, a new strategy for nuclease assay has been developed using conjugated polymer and DNA/intercalating dye complex. The discrimination of DNA before or after digestion by nuclease denotes the universal application of the approach, in which either dsDNA or ssDNA substrates could be used for detecting nuclease activity. This method can be extended to most nucleases by simply changing the substrate DNA. The present method is label-free, rapid, and highly sensitive, with a detection limit much better or at least comparable to previous reports. In addition, this assay is easy to implement for visual detection with the assistance of a UV transilluminator. We reason that a similar strategy can be adopted for the detection of other analytes.