The sensing mechanism of a hemicyanine-based fluorescence probe for reactive oxidative species was investigated using TDDFT method. Geometries of probe and oxidation product were optimized at the M06/TZVP/SMD level. Vertical excitation energies of probe molecule corresponded well with experimental UV–Vis bands. The planar ground-state geometry and vertical excited-state geometry of probe suggested a TICT process that led to the fluorescence quenching. By contrast, the oxidation product would not go through the TICT process due to long-range substituent effect, thus, displaying strong fluorescence. The analysis results of their frontier orbitals and potential energy scan also confirmed the above conclusions.