Stimuli-responsive supramolecular host assisted guest aggregation has attracted tremendous attention, of late, because of its various technological applications. However, a large majority of them involve anionic supramolecular hosts such as p-sulfonato calixarenes, sulfated cyclodextrin and so on, in combination with cationic guests. On the other hand, the phenomenon of host-assisted aggregation of anionic guests with cationic supramolecular hosts, which can significantly broaden the scope of this exciting field, has rarely been reported in the literature. In the present work, we report, host assisted aggregation of an anionic organic probe molecule, pyrene tetrasulfonate (PTS), in the presence of a cationic cyclodextrin derivative, amino-β-CD (AβCD), which has been studied, in detail, using various spectroscopic techniques including UV–Vis spectroscopy, fluorescence spectroscopy, time-resolved spectroscopy, and molecular docking calculations. The effect of ionic strength of the medium on the photophysical properties of PTS-AβCD complex indicates that electrostatic interaction is the predominant force in assisting the formation of PTS-AβCD complex. The monomer-aggregate equilibrium of PTS in the present system is also found to be extremely sensitive towards external stimuli like temperature, and pH of the medium. Finally, the PTS-AβCD assembly has been used for sensing an important charged bio-analyte, adenosine triphosphate (ATP), demonstrating the applicability of the present host-guest system.