Abstract: The sensing behaviors of a rhodamine derivative RhB-NSal bearing a –NO2 group for Cu2+ in dry acetonitrile and aqueous acetate buffer media were investigated. In each medium, significant absorption and fluorescence enhancements accompanied by an instant color change were observed for RhB-NSal upon addition of Cu2+. RhB-NSal binds with Cu2+ forming a 1:1 stoichiometric complex with an association constant of 6.72 × 104 and 4.23 × 104 L/mol, respectively. RhB-NSal displayed high selectivity for Cu2+ over other ions except that Fe3+ and Bi3+ ions induce a little interference. In dry acetonitrile, Cu2+ results in remarkable spectroscopic enhancements of RhB-NSal with a detection limit of 0.49 μmol/L for Cu2+, exhibiting higher sensitivity than that of an analogue bearing no substituent on its phenol ring. In aqueous solution, RhB-NSal likewise displays a high selectivity but a lower sensitivity for Cu2+ than that in acetonitrile, with a detection limit of 14.98 μmol/L, still more sensitive than RhB-Sal in absorption. By virtue of these properties, RhB-NSal could be used as a promising candidate of an “Off-On” fluorescent and colorimetric chemosensor for Cu2+ in different media.