Lipid droplets (LDs) are crucial dynamic organelles within cells that play an indispensable role in cellular energy supply, membrane formation, and signal transduction. Therefore, detecting and tracking LDs with high selectivity and resolution is particularly important. In this study, we describe a “trident” anchoring strategy (based on polarity, lipophilicity, and viscosity sensitivity) to achieve highly specific and dynamic imaging of LDs. A series of carbazole-substituted fluorescent protein (Car-FP) mimics conjugated with different electron-nature substituents (A–H) were synthesized and characterized. Spectroscopic properties indicate that the electron-donating triphenylamine (TPA)-conjugated H exhibits high sensitivity to polarity, viscosity response, and lipophilicity. Co-localization experiments in cells also demonstrate that H selectively localizes at LDs in living cells, displaying a bright yellow fluorescence signal (λex = 488 nm; λem = 577 nm) with a larger Stokes shift (89 nm) than the commercial LD dye bodipy 493/503 (10 nm). Super-resolution fluorescence imaging (SIM) and lifetime imaging (FLIM) are employed to visualize the distribution and aggregation in cell models of nonalcoholic fatty liver disease as well as zebrafish and mice models, demonstrating the potential applications of H in LD biology research and diagnosis of related diseases.