Self-assembled multimolecular aggregates, such as vesicles, have earned tremendous attention for their applications as model membranes and drug delivery systems. Over the past decades, enormous efforts have been dedicated to the development of stable and biocompatible vesicles that form spontaneously in aqueous solution. With the aim of preparing highly stable vesicles, we herein report the physicochemical characterization of a novel cholesterol-based chiral surfactant with L-alanine headgroup. Different techniques, such as surface tensiometry, fluorescence spectroscopy, dynamic light scattering, UV-vis spectroscopy, transmission electron microscopy, and confocal fluorescence microscopy were employed to investigate the self-assembly properties of the aforementioned single-tailed steroidal surfactant in aqueous solution. The surfactant molecule is weakly surface-active, but self-assembles to form unilamellar vesicles facilitated by the strong hydrophobic association of the cholesterol moieties, above a very low critical aggregation concentration. The vesicles are fairly stable with respect to aging, temperature, and pH of the aqueous medium. Additionally, the vesicles were found to fuse together, leading to large unilamellar vesicles. The intervesicular fusion pertaining to high stability of the vesicles could be ascribed to large hydrophobic interactions among steroidal skeletons. Furthermore, the interaction of the vesicles with human serum albumin is also investigated.