Intracellular lipid storage and regulation occur in lipid droplets, which are of great significance to the physiological activities of cells. Herein, a lipid droplet-specific fluorescence probe (lip-YB) with a high quantum yield (QYlip-YB = 73.28%), excellent photostability, and quickly polarity sensitivity was constructed successfully. Interestingly, lip-YB exhibited remarkable two-photon (TP) characteristics, which first realized real-time monitoring of the lipid droplet multidynamics process, diagnosing nonalcoholic fatty liver disease (NAFLD) and inflammation in living mice via TP fluorescence imaging. It is found that the as-prepared lip-YB provides a new avenue to design lipid droplet-specific imaging probes, clarifies its roles and mechanisms in cell metabolism, and can timely intervene in lipid droplet-related diseases during various physiological and pathological processes.