A rhodamine-based chemosensor was synthesized and found to selectively bind ferric ions over other metal ions (Na+, K+, Ca2+, Mg2+, Fe2+, Zn2+, Cd2+, Co2+, Hg2+ Cr3+, Al3+) in an organic-aqueous mixture (CH3CN-MES). Upon addition of ferric ions, the spirolactam ring opens, producing a visual color change and a fluorescence intensity increase, i.e. a “turn on” optical response at 577 nm is observed. The chemosensor coordinates to ferric ions in 1:1 stoichiometry with a calculated K a = 3.5 × 104 mol⋅dm−3 by fluorescence spectroscopy and a LoD of 27 ppb. The chemosensor was reversible upon addition of the Fe3+ chelator desferrioxamine. One- and two-dimensional NMR experiments with Al3+ ions aided in understanding of the coordination environment of the ferric ion with the chemosensor, which were confirmed by molecular modeling calculations. X-ray quality crystals of the chemosensor were obtained, and the solid-state structure is reported. Confocal microscopy was used to detect free ferric ions in Staphylococcus aureus.