The mechanism of β-galactosidase detection probes ESIPT-GAL and ESIPT-OH have been investigated. The torsional potential energy curve proves the planar characteristics of the system. Combined with frontier molecular orbital and “hole-electron” analysis, there are apparent planar intramolecular charge transfer (PICT) characteristics in ESIPT-GAL and ESIPT-OH. The barrier-free intramolecular proton transfer explained the single fluorescence. Furthermore, the mechanism of ESIPT-OH fluorescent probe is the cooperation of excited-state intramolecular proton transfer (ESIPT) and PICT. The relationship between the singlet and triplet states combined with the spin coupling constant reveals that the weak fluorescence characteristic of ESIPT-GAL is caused by the intersystem crossing.