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A new coumarin-acridone fluorescent probe S was designed and synthesized, and the structure was confirmed with ¹H/¹³C NMR spectrometry, single-crystal X-ray diffraction, and high-resolution mass spectrometry. This probe has high sensitivity and selectivity for Fe³⁺ over other testing metal ions at 420 or 436 nm in acetonitrile–MOPS (3-Morpholinopropanesulfonic Acid) buffer solution (20.0 μM, pH = 6.9, 8:2 (<i>v</i>/<i>v</i>)). Under physiological conditions, the probe displayed satisfying time stability with a detection limit of 1.77 µM. In addition, probe S was successfully used to detect intracellular iron changes through a fluorescence-off mode, and the imaging results of cells and zebrafish confirmed their low cytotoxicity and satisfactory cell membrane permeability, as well as their potential biological applications.