Peroxynitrite (OONO-) is profoundly implicated in health and disease. The physiological and pathological outcome of OONO- is related to its local Concentration, and hence, a reliable OONO- assay is highly desired. We have developed a FRET-based small molecule fluorescent probe (PNCy3Cy5), harnessing the differential reactivity of Cy3 and Cy5 toward OONO- by fine-tuning. It exhibits high detection sensitivity and yields a ratiometric fluorescent signal. We have exemplified that it can be applied in semiquantitative determination of OONO- in living cells. Notably, it specifically localizes in mitochondria where endogenous OONO- is predominantly generated. Thus, PNCy3Cy5 is a promising molecular tool for peroxynitrite biology.