Thiazole orange (TO), an intercalating dye, is integrated into cationic poly(fluorene-alt-phenylene) (PFP) to develop a macromolecular multicolor probe (PFPTO) for double-stranded DNA (dsDNA) detection. This polymer design not only takes advantage of the high affinity between TO and dsDNA to realize dsDNA recognition in biological media, but also brings into play the light-harvesting feature of conjugated polymers to amplify the signal output of TO in situ. PFPTO differentiates dsDNA from single-stranded DNA (ssDNA) more effectively upon excitation of the conjugated backbone relative to that upon direct excitation of TO as a result of efficient fluorescence resonance energy transfer from the polymer backbone to the intercalated TO. In the presence of dsDNA, energy transfer within PFPTO is more efficient as compared to that for free TO/PFP system, which leads to better dsDNA discriminability for PFPTO in contrast to that for TO/PFP. The distinguishable fluorescent color for PFPTO solutions in the presence of dsDNA allows naked-eye detection of dsDNA with the assistance of a hand- held UV lamp. significant advantage of this macromolecular fluorescent probe is that naked-eye detection of label-free dsDNA can be performed in biological media in real-time.