This article presents results of spectroscopic studies applied to a selected 1,3,4-thiadiazole compound, 4-[5- (naphthalen-1-ylmethyl)-1,3,4-thiadiazol-2-yl]benzene-1,3-diol (NTBD), in micellar systems. In this study the non- ionic surfactant Triton X-100 was preferred to the analysis. Detailed spectroscopic studies were carried out using absorption and fluorescence spectroscopy, resonance light scattering (RLS), as well as dynamic light scattering (DLS). Time-resolved fluorescence lifetime measurements supplemented the spectroscopic results. Moreover, monomeric structures and aggregated forms in the aforementioned 1,3,4-thiadiazole analogue were character- ized using X-ray crystallographic techniques. The steady-state fluorescence measurements revealed very inter- esting effects in the emission bands, where NTBD displays only a single fluorescence emission in methanol but shows pronounced dual fluorescence in TX-100 micellar solutions. These emissions occur at ~375 nm and ~ 495 nm, corresponding to its enol* and keto* tautomer’s which in our previous paper were related to the excited-state intramolecular proton transfer (ESIPT) effect. This dual emission indicates that surfactant-driven aggregation strongly promotes ESIPT (aggregation-induced emission, AIE). Surface Plasmon Resonance (SPR) and Electro- chemical impedance spectroscopy (EIS) measurements confirmed NTBD–micelle interactions, and X-ray crys- tallography revealed aggregated NTBD structures. Moreover, the binding constants (Kb) and the thermodynamic parameter, such as the change in Gibbs energy (ΔGb) have been determined for the NTBD-Triton X-100 system. Overall, NTBD’s unique AIE-enhanced ESIPT behavior highlights its novelty and suggests its potential as a hydrophobicity-sensitive fluorescent probe in biological and pharmaceutical research.