The production of peroxynitrite (ONOO–) in situations of oxidative stress and inflammation has a profound impact on cellular damage, inflammatory responses, and immune regulation. Thus, the advancement of highly sensitive and selective techniques to detect ONOO– is of great significance. These methods are crucial for gaining insights into disease mechanisms, improving early disease diagnosis, and enhancing treatment effectiveness. In this study, we have designed and synthesized a novel fluorescent probe, NR-Pro, specifically for the selective detection of ONOO–. The probe incorporates a 2-hydroxy Nile red derivative (NR-OH) that is organically bound to a 4,4'-azabediyldiphenol group via an alkyl chain. Upon interaction with ONOO–, the probe releases the near-infrared dye NR-OH, leading to a significant “OFF-ON” fluorescence signal response at 658 nm. The mechanism involves the initial attack of ONOO– on the 4,4'-aza-diyl diphenol group, resulting in the formation of an alkylamine-modified Nile red derivative and the release of two 1,4-benzoquinone molecules. Subsequently, the alkylamine group undergoes further oxidation by ONOO–, leading to the liberation of the near-infrared fluorophore NR-OH and acrolein through a 1,3-elimination reaction with the involvement of water. Experimental investigations have demonstrated that the probe exhibits a favorable linear relationship with ONOO– concentration in the range of 0 to 10 μmol/L, with an exceptionally low detection limit of 17.7 nmol/L. Furthermore, the fluorescence emission of NR-Pro remained unaffected by various biological species, including reactive oxygen species (ROSs), metal ions, enzymes, anions, and amino acids. NR-Pro also exhibited minimal cytotoxicity and very high photostability, suggesting its potential suitability for cell imaging applications. Moreover, our study confirms the remarkable imaging capability of the NR-Pro probe in detecting both exogenous and endogenous ONOO– in RAW264.7 cells. These findings present novel insights for the early diagnosis and treatment of diseases, paving the way for potential advancements in the field.