A benzoxazole-based fluorescent probe 4-(benzo[d]oxazol-2-yl)-2-((bis(2-(ethylthio)ethyl)amino)methyl)phenol (ZY1) was designed and synthesized, and characterized by mass spectrometry (MS), nuclear magnetic resonance (NMR) and X-ray single crystal diffraction, etc. ZY1 was prepared by Mannich reaction, and its pale yellow crystals suitable for X-ray diffraction studies were obtained by slow evaporation of an ethyl acetate solution at room temperature over a period of a few days. The results showed that ZY1 exhibited good sensitivity and selectivity to Hg2+. The fluorescence of ZY1 was quenched dramatically in the presence of Hg2+, and it remained nearly constant in the range of pH 3.0～9.0. Other metal ions, such as K+, Ca2+, Na+, Mg2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Pb2+, induced negligible fluorescence quenching for ZY1 under the same conditions. When the concentration of Hg2+ was increased from 0 to 30 μmol/L, the fluorescence of ZY1 (10 μmol/L) quenched gradually. Binding analysis using the method of continuous variations (Job's plot) established that the stoichiometry of ZY1-Hg2+ complex was estimated to be 1:1. The fluorescence intensity varied almost linearly vs. the concentration of Hg2+ (0.5～4.8 μmol/L), and the detection limit of Hg2+ was estimated to be 0.08 μmol/L. Moreover, ZY1 was used for the visual detection of Hg2+ and applied to image intracelluar Hg2+ in living HeLa cells, indicating that it could be a useful tool in bioanalytical applications.