In view of the application prospects in biomedicine of (E)-1-(4-(diethyla-mino)-2-hydroxybenzylidene)-4,4-dimethylthiosemicarbazide (DAHTS), the behavior of excited-state dynamics and photophysical properties were studied using the density functional theory/time-dependent density functional theory method. A series of studies indicated that the intramolecular hydrogen-bond (IHB) intensity of DAHTS was enhanced after photoexcitation. This was conducive to promoting the excited-state intramolecular proton-transfer (ESIPT) process. Combining the analysis of the IHB and hole–electron, it revealed that the molecule underwent both the ESIPT process and the twisted charge-transfer (TICT) process. Relying on exploration of the potential energy surface, it was proposed that the different competitive mechanisms between the ESIPT and TICT processes were regulated by solvent polarity. In acetonitrile (ACN) solvent, the ESIPT process occurred first, and the TICT process occurred later. In contrast, in the CYH solvent, the molecule first underwent the TICT process and then the ESIPT process. Furthermore, we raised the possibility that the TICT behavior was the cause of weak fluorescence emission for the DAHTS in CYH and ACN solvents. By the dimer correlation analysis, the corresponding components of triple fluorescence emission were clearly assigned, corresponding to the monomer, dimer, and ESIPT isomer in turn. Our work precisely elucidated the photophysical mechanism of DAHTS and the attribution of the triple fluorescence emission components, which provided valuable guidance for the development and regulation of bioactive fluorescence probes with multiband and multicolor emission characteristics.