By modifying the structure of pyridoxal phosphate, an “on–off” rhodamine fluorescent probe LPDQ with a new structure was synthesized. NMR, IR, and mass spectrometries characterized the structures of the probes. The LPDQ–Zn2+ system had been successfully constructed by the characteristic absorption peaks of the ultraviolet spectrum, and the experiments were carried out to investigate the ion fluorescence selectivity, cell imaging, reversibility, ion concentration, linear range, precision, and accuracy. The LPDQ reached the detection limit of 0.8 × 10−6 mol/l at 1 × 10−5–8.5 × 10−5 mol/l. The content of Zn2+ in the zinc gluconate sample was determined. According to the experimental data, the fluorescent probe LPDQ in this experiment has good performance and can be used to detect Zn2+ in water and oil paintings.