Herein, we present the rational design, synthesis, characterization, and biological applications of a new rhodamine-based fluorescent probe, HKYellow, for the detection and molecular imaging of peroxynitrite, an important highly reactive oxidant involved in a variety of physiological and pathological processes. HKYellow was rationally designed on the basis of the efficient fluorescence quenching effect of the N-phenyl group to the rhodamine core and a peroxynitrite-triggered N-dearylation reaction to achieve a sensitive and selective fluorescence turn-on response toward peroxynitrite in chemical and biological levels. This probe has been thoroughly evaluated for the robust imaging of peroxynitrite in live cells and tissues. By utilizing HKYellow, we provide the first visual evidence that peroxynitrite is generated in mouse liver tissues under an acute alcohol binge or ischemic-reperfusion condition. This probe should be a powerful molecular imaging tool for interrogating the complex biological roles of peroxynitrite under various biological settings.