Herein, a 2-furyl-3-hydroxychromone-based deoxyuridine analogue was synthesized and incorporated into DNA. Its ESIPT mechanism, investigated by steady-state and time-resolved spectroscopy, was found to be highly sensitive to hydration through its ratiometric response. As a result, this analogue demonstrated a unique on-off dual emissive behavior which allows monitoring DNA hybridization as well as discriminating matched from mismatched duplexes, and B from A conformations. © 2016 The Royal Society of Chemistry.