We present a strategy to generate a light-up fluorophore-aptamer pair. The strategy was based on a modification of a conventional DNA-staining dye to suppress its affinity to the original targets, and subsequent re-selection of aptamers that would bind to the modified dye. Along this line, we prepared an environmental polarity-sensitive Hoechst derivative with low affinity to the usual AT-rich dsDNA targets. DNA aptamers, in vitro selected from a random pool, worked as a trigger to enhance the fluorescence of an otherwise nonfluorescent Hoechst derivative.