Dual-function chemosensors that combine the capability of colorimetric and fluorimetric detection of Cu2+ are still relatively rare. Herein, we report that a 3-hydroxyflavone derivative (E)-2-(4-(dimethylamino)styryl)-3-hydroxy-4H-chromen-4-one (4), which is a red-emitting fluorophore, could serve as a reversible colorimetric and fluorescence “turn-off” chemosensor for the detection of Cu2+. Upon addition of Cu2+ to 4 in neutral aqueous solution, a dramatic color change from yellow to purple-red was clearly observed, and its fluorescence was markedly quenched, which was attributed to the complexation between the chemosensor and Cu2+. Conditions of the sensing process had been optimized, and the sensing studies were performed in a solution of ethanol/phosphate buffer saline (v/v = 3:7, pH = 7.0). The sensing system exhibited high selectivity towards Cu2+. The limit of naked eye detection of Cu2+ was determined at 8 × 10−6 mol/L, whereas the fluorescence titration experiment showed a detection limit at 5.7 × 10−7 mol/L. The complexation between 4 and Cu2+ was reversible, and the binding constant was found to be 3.2 × 104 M−1. Moreover, bioimaging experiments showed that 4 could penetrate the cell membrane and respond to the intracellular changes of Cu2+ within living cells, which indicated its potential for biological applications.