Fe3+ is essential for humans, and its deficiency or excess can be harmful to human health; thus, it is crucial to detect Fe3+. Herein, a novel 1,8-naphthylimide rhodamine-based fluorescent probe (NA-RhBEA) was prepared from rhodamine B, anhydrous ethylenediamine, and 1,8-naphthoic anhydride. This fluorescent probe complexes Fe3+ with N and O on the carboxyl groups of its spironolactam structure and part of the 1,8-naphthalenedicarboxylic anhydride structure, which results in spironolactam ring-opening and fluorescence. NA-RhBEA has high selectivity for Fe3+ in ethanol/buffer solution (4 : 1, v/v), and fluorescence is detected at an excitation wavelength λEX = 500 nm, an absorption peak appears at 585 nm, and a significant color change appears. The effect of the fluorescence intensity of Fe3+ under a series of different concentration conditions was investigated, and it was concluded that the fluorescence intensity increased with increasing Fe3+ concentration in the range of 0-500 μmol, and its detection limit was 0.84 μmol L-1. In addition, we explored the detection ability of NA-RhBEA in solutions with different pH values, mixed metal ions, and different solvents, and the results showed that the fluorescent sensor also has good anti-interference properties and some practical applicability.