A novel rhodamine-based multi-ion fluorescent sensor, RGN, was designed and synthesized for the highly selective detection of mercury ions (Hg2+) in ethanol and water systems, as well as trivalent cations (Fe3+, Al3+, and Cr3+) in acetonitrile and water systems using a two-step Schiff base reaction method. Nuclear magnetic titration experiments and theoretical calculations demonstrated that the sensor achieved the detection of the aforementioned metal ions through the fluorescence turn-on phenomenon induced by lactam ring-opening. Density functional theory (DFT) calculation results showed decreased HOMO-LUMO energy gaps and increased dipole moments, indicating the effective coordination of the sensor with the corresponding metal ions to form more stable complexes, thereby achieving detection objectives. Furthermore, the fluorescence turn-on sensor RGN exhibited relatively low detection limits, with limits of detection (LOD) for Fe3+, Al3+, Cr3+, and Hg2+ being 10.20 nM, 14.66 nM, 58.78 nM, and 73.33 nM, respectively. Finally, practical applications of sensor RGN in environmental water samples, L929 cells, and zebrafish were demonstrated, indicating its potential for detecting and tracking Fe3+, Al3+, Cr3+, and Hg2+ in environmental samples and biological systems, with prospects for biomedical applications in the diagnosis and treatment of heavy metal ion-induced diseases.