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The fluorescent probe, GXY-ADP-2, with xanthene structure as the fluorescent core was designed and prepared for the selective detection of peroxynitrite (ONOO−). ONOO− can be produced endogenously and exogenously and is a strong oxidant with a short half-life. Oxidative modifications of biomolecules, that can be attributed to the formation of ONOO−, occur in the reactions of biomolecules with secondary ONOO−-derived radical oxidants. Therefore, it is very important to develop a specific fluorescent probe for detecting ONOO− to monitor oxidative stress state. The excitation wavelength and emission wavelength of the probe are 689 nm and 739 nm respectively. In the process of co-incubation with ONOO−, generate a new substance with two internal conjugated structures through a special reaction mechanism, one giving the fluorescence with the excitation wavelength of 347 nm and the emission wavelength of 484 nm with the detection limit of 0.12 μM, and the other that with the excitation wavelength of 433 nm and the emission wavelength of 583 nm with the detection limit of 0.077 μM. The linear dynamic range of the probe is 0–5 μM. Its response is not affected by the other reactive oxygen species, thus can sensitively detect ONOO−. In bioimaging experiments with HepG2 cells, the green and blue cell fluorescence signals (583 nm and 433 nm, respectively) were increased, while the red one (739 nm) was significantly reduced, under lipopolysaccharide (LPS) induced oxidative stress, proving that the probe could sensitively detect ONOO− in living cells. This work provides a new tool for the dynamic changes of ONOO− and oxidative stress processes in biological systems. [Display omitted] • A new fluorescent probe, GXY-ADP-2, was developed for detection of ONOO−. • GXY-ADP-2 exhibited high specificity and sensitivity to ONOO−. • GXY-ADP-2 is a ratiometric fluorescent probe with three-channel fluorescence signal changed. • GXY-ADP-2 can detect the dynamic change of ONOO− in living cells. [ABSTRACT FROM AUTHOR]