In this study, the far-red-emitting fluorescence probe 1, containing a rhodamine derivative and a hydrazide reactive group, was developed for peroxynitrite detection and imaging. This probe, which is cell permeable and shows high sensitivity and selectivity in fluorometric detection of peroxynitrite over other ROS/RNS, was successfully utilized to detect exogenous and endogenous peroxynitrite in HeLa and RAW 264.7 cells, respectively. More importantly, 1 can also be used to detect endogenous peroxynitrite generated in Pseudomonas aeruginosa (PAO1)-infected mouse bone marrow-derived neutrophils. We anticipate that the new probe will serve as a powerful molecular imaging tool in investigations of the role(s) played by peroxynitrite in a variety of physiological and pathological contexts.