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Reactive oxygen species (ROS) are pivotal signaling molecules that control a variety of physiological functions. As a member of the ROS family, peroxynitrite (ONOO⁻) possesses strong oxidation and nitrification abilities. Abnormally elevated levels of ONOO⁻ can lead to cellular oxidative stress, which may cause several diseases. In this work, based on the rhodamine fluorophore, we designed and synthesized a novel small-molecule fluorescent probe (<b>DH-1</b>) for ONOO⁻. Upon reaction with ONOO⁻, <b>DH-1</b> exhibited a significant fluorescence signal enhancement (approximately 34-fold). Moreover, <b>DH-1</b> showed an excellent mitochondria-targeting capability. Confocal fluorescence imaging validated its ability to detect ONOO⁻ changes in HeLa and RAW264.7 cells. Notably, we observed the ONOO⁻ generation during the ferroptosis process by taking advantage of the probe. <b>DH-1</b> displayed good biocompatibility, facile synthesis, and high selectivity, and may have potential applications in the study of ONOO⁻-associated diseases in biosystems.