A simple and new fluorescent and colorimetric probe bearing 7-nitrobenz-2-oxa-1,3-diazole and 4-maleimidophenol fragments for biothiols was designed and synthesized. The probe itself showed almost no background fluorescence (Φ_F < 1 × 10⁻⁴) and displayed fluorescence turn-on response with selectivity for thiols over other relevant biological species in aqueous solutions. In addition, the probe exhibited 110 nm red-shifted absorption spectra accompanied by color changes which could be obviously distinguished by the naked eye. Furthermore, the probe showed high sensitivity towards thiols with a detection limit of 1.2 × 10⁻⁷ M (S/N = 3) and the mechanism regarding the optical responses of the probe to thiols was explained by density function theory (DFT) calculations. Finally, the probe has been successfully used to image thiols in HeLa cells.