It is well reported that sugar molecules provide different types of stabilization to biomembranes both in vitro as well as in vivo. In the present article, our focus is to investigate the interactions of two sugar molecules (sucrose and sucralose) with the lamellar structures of aerosol OT (AOT). We have attempted the structural characterization of the lamellae in the presence and absence of sugars with the aid of dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), Fourier transformed infrared spectroscopy (FTIR), polarized optical microscopy (POM), and cryogenic-transmission electron microscopic (cryo-TEM) techniques. In this study, we have chosen three Coumarin dyes, which show a wide variation of hydrophobicity and performed a combination of steady-state and time-resolved fluorescence measurements to unveil the inner detail of the location of the sugars' and their specific interactions with the lamellar structures. Our study reveals that sucrose molecules are present in the interfacial region with a major population whereas the most probable location of sucralose is the interior of the AOT bilayer. Therefore, sucralose molecules probably penetrate the bilayer by decreasing the efficient packing of AOT. The important essence of this study is the location and the interactions of sucralose with the lamellae which may provide a future direction to the transportation of the drug molecules in the biomembrane.