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β-Glucuronidase (GLU) as a vital factor in enterohepatic circulation and drug-inducing enteropathy has been given more and more attention in recent years. In this study, an off-on near-infrared (NIR) fluorescent probe (DDAO-glu) for selectively and sensitively sensing GLU was developed on the basis of its substrate preference. DDAO-glu can rapidly and selectively respond to bacterial GLU under physiological conditions for detecting the real-time intestinal GLU bioactivity of complex biological systems such as human feces in clinic. Meantime, DDAO-glu has been successfully applied for visualization of endogenous GLU in bacterial biofilm, thallus, and even in vivo. Using this NIR probe, we successfully visualized the real distribution of intestinal GLU in the enterohepatic circulation. Furthermore, a high-throughput screening method was successfully established by our probe, and a potent natural inhibitor of GLU was identified as (-)-epicatechin-3-gallate (ECG) for effectively preventing NSAIDs-inducing enteropathy in vivo. DDAO-glu could serve as a powerful tool for exploring real physical functions of intestinal GLU in enterohepatic circulation, under physiological and pathological contexts, and developing the novel inhibitors of GLU to therapy acute drug-inducing enteropathy in clinic.