Development of a fluorescent probe for activity‐based sensing of activity of alcohol dehydrogenase, a key enzyme in ethanol biooxidation, is reported. A caged coumarin reporter is released upon the selective oxidation by the enzyme with a strong, >60‐fold emission enhancement. The probe has a low cytotoxicity and has been applied in visualising alcohol dehydrogenase activity in HepG2, A549 and HEK293T cells, demonstrating its potential as a convenient, easy‐to‐use bioanalytical tools in unveiling the roles of the enzyme in alcohol metabolism.
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