Hydrogen sulfide (H2S) and nitroreductases (NTR) are key biological reductants, with H2S acting as a key signaling molecule and NTR reducing nitro groups to amines in cellular metabolism. Visualizing these reducing components, often overexpressed in cancer cells or bacterial environments, is crucial for metabolic understanding. Existing fluorescent probes detect these individually, but simultaneous detection methods are unexplored. We designed probe 1, which shows high fluorescence only when both NTR and H2S are overexpressed. The probe transformed into benzothiazole‐fused coumarin by NTR, exhibiting fluorescence, and shows a stronger signal in the presence of both NTR and H2S, making it a potential tool for detecting their simultaneous overexpression.