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Peroxynitrite (ONOO - ), a kind of reactive oxygen species, plays an indispensable role in many physiological processes. The stability and reactivity of ONOO - are significantly affected by the pH of the environment. A novel fluorescent probe RN-NA that can simultaneously respond to ONOO - and pH was proposed and constructed based on a rational-designed multifunctional fluorescence resonance energy transfer (FRET) platform. The RN-NA probe exhibited a remarkably different fluorescence change in response to ONOO - and pH. The fluorescence signals at 525 and 710 nm increased about 4-fold with a pH change from 8.0 to 3.0. The changes in fluorescence at 525 nm are mainly attributed to photo-induced electron transfer, and the fluorescence enhancement at 710 nm was mainly due to acid-induced open-closed circulation. In the presence of ONOO - , the fluorescence at 525 nm increased 5-fold, while the fluorescence at 710 nm was almost completely diminished. Up to 70-fold fluorescence enhancement was observed in the ratiometric channel F 525 / F 710 . In the cell imaging experiment, the intracellular pH was adjusted using H + /K + ionophore and nigericin, and the endogenous ONOO - was generated by lipopolysaccharide (LPS) and γ-interferon (IFN-γ). The RN-NA probe can respond to cellular pH and endogenous ONOO - with remarkable fluorescence changes in both red/green and ratiometric channels.