Disclosed are a fluorescent probe for detecting a nitroreductase, a preparation method therefor and the use thereof in an enzymatic reaction, wherein same fall within the technical field of industrial analysis and detection. The fluorescent probe is 3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-yl)vinyl)quinoline-1-bromo)propane-1-sulfonate. The probe compound of the present invention enhances the hydrophilicity thereof by introducing hydrophilic groups such as sulfonates and quinoline salts, and performs 1,6-rearrangement and elimination reactions under the catalysis of a nitroreductase (NTR) to generate a hydroxyl, and realizes the detection and analysis of NTR in an enzymatic reaction by means of fluorescence variations triggered by an intramolecular charge transfer (ICT) effect. The method has the advantages of being simple to prepare, a high yield, and being suitable for detecting the content of a high-concentration enzyme in an enzymatic reaction, and also exhibits great application prospects in the enzyme detection field for enzymatic reaction systems in the field of chemistry.
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