A new Rhodamine B derivative RBMAB, namely 3-amino-N-(2-(3',6'-bis (diethylamino)-3-oxospiro[isoindoline-1,9'-xanthen]-2-yl) ethyl)benzamide, was designed, synthesized and structurally characterized to develop a chemosensor. The studies show that RBMAB exhibits high selectivity toward Fe 3+ among many other metal cations in an ethanol-H 2 O (3:2, v/v, PBS buffer, 1.2 mmol L -1 , pH 7.2) solution. It has a low detection limit of 0.021μm. Fluorescence microscopy experiments further demonstrate that RBMAB can be used as a fluorescent probe to detect Fe 3+ in living cells.