Small-molecule-based fluorescent chemosensors provide powerful tools for analytical chemistry. However, their organic essence often "cursed" them for aggregation-caused quenching (ACQ) in an aqueous context. Albeit the praxis of the disaggregation-induced emission (DIE) strategy as a potential solution, it still awaited improvement due to the uncontrollability of the aggregation/segregation process. To address this issue, herein, we supposed that sulfur substitution on a molecule could serve as a promising strategy to achieve an evolved ACQ-DIE probe. To prove this concept, a precursor G-quadruplex (G4) ligand CQ was modified to get its thionation version CTQ. Strikingly, CTQ exhibited more arranged aqueous segregation behavior, as compared with CQ, and therefore enhanced fluorescence performance. Our research, in the meantime, manifested that CTQ remained to possess favorable G4 selectivity, whereby it could function as an evolved probe for more accurate in vitro G4-related assays, specifically, a classification assay for distinguishing virus variants.