In the present paper, a new spirobenzopyran derivative was synthesized and applied in simultaneously colorimetric and fluorescence ratiometric detections of Cu2+. In contrast to the virtually photochromic character of the common spirobenzopyrans in most organic solvents, this spirobenzopyran is colorless in organic aqueous solution even irradiating by ultraviolet light. The formation of red merocyanine in an ethanol-aqueous solution is only induced by Cu2+ coordination. Furthermore, the closed form of the spirobenzopyran is highly fluorescent. Upon complexation with Cu2+, it displays not only decreasing in the initial fluorescence emission band but also appearing in a new emission at long wavelength. Thus, the Cu2+ quantitative measure can be achieved by fluorescence ratiometry. With the optimum conditions described, the Cu2+ concentration can be determined from 5.13 x 10(-7) M to 3.81 x 10(-4) with a detection limit of 1.06 x 10(-7) M. Both the color and the fluorescence changes of the spirobenzopyran are extremely specific for Cu2+ over biologically relevant substrates, which meet the selectivity requirements for biomedical application. Serum divalent copper values were determined using this spirobenzopyran, which fell into the normal range of the content reported in the literature and were in good agreement with those obtained by atomic absorption spectroscopy. The combined data from fluorescence titrations and 1H NMR measurements indicate that the new emission of the spirobenzopyran generated by Cu2+ is the result of the metal-induced ring opening and conformation restriction by Cu2+ liganding with the opened merocyanine form and the subsequent reduction of the intramolecular charge transfer of the merocyanine.