The farnesoid X receptor (FXR) regulates key physiological processes, such as bile acid homeostasis and lipid metabolism, making it an important target for drug discovery. However, the overactivation of FXR often leads to adverse effects. This study presents the development of a novel fluorescent probe utilizing the computer-aided drug design (CADD) approach to optimize linkers between more potent warhead and FITC fluorescent groups. The probes were designed and assessed via molecular dynamics simulations, and four were selected for synthesis to be evaluated in in vitro biochemical assays. Among these, ABDS-2 exhibited high sensitivity and stability, which demonstrated satisfactory validation in high-throughput screening assays. Furthermore, ABDS-2 facilitated real-time bioimaging to monitor FXR homeostasis at the cellular level, providing spatially resolved insights into molecular interactions critical for cellular function studies. This research underscores the efficiency of CADD in probe design and positions ABDS-2 as a valuable chemical tool for in vitro assays and cellular-level bioimaging.