Bile salts (BS) are naturally occurring steroidal biosurfactants. The ease of functionalization of BSs has boosted their use as inexpensive building blocks for the fabrication of a broad set of value-added soft functional materials. In the present work, three fluorescent bile acid (FBA) derivatives have been synthesized by conjugating anthracene at the side chain of lithocholic acid, deoxycholic acid, and cholic acid to understand the effect of the nature of the steroid nucleus on their physicochemical properties. In an aqueous medium, the FBAs showed a strong supramolecular aggregation propensity, even in the micromolar concentration range, which is in contrast to their BS analogues that form micelles mostly in the millimolar range. The FBA aggregation leads to a prearranged geometry in the ground state with a favorable orientation of anthracene units for excimer formation on excitation, leading to supramolecular aggregation-induced enhanced emission (AIEE). A detailed investigation reveals the pivotal role of the steroidal skeleton in their aggregation propensity and optical behavior. The FBA assemblies, with ordered structures plus anthracene being a part of their building blocks, are endowed with interesting properties different from those in dilute organic media, which makes them extremely attractive for diverse applications, e.g., as potential drug carriers owing to their ability to serve as efficient hosts for the protective encapsulation of hydrophobic guests; as membrane probes and bioimaging agents due to their efficient membrane permeability and cell-imaging ability; and as system probes because of their remarkable sensitivity toward the aggregation process of natural bile salts in the aqueous medium. Therefore, the present study not only enhances the fundamental understanding of this unique class of amphiphiles but also opens new prospects in tailoring novel self-assembled soft functional materials. Moreover, it offers a benchmark for developing BS-based fluorescent derivatives with unique photophysical characteristics for applications as potential bioprobes.