The development of sensitive methods for imaging endogenous peroxynitrite (ONOO−) in the mitochondria of living cells is very important to disclose its diverse physiological and pathological functions. In this paper, we have described a simple, mitochondria-targeted far-red fluorescent probe Mito-PN for selectively detecting ONOO− even though in the presence of higher concentration (1mM) other biologically important substances. The experimental results indicated that probe Mito-PN could quantitatively determine ONOO− in the range of 0.5–8μM with the detection limit of 17nM. In addition, probe Mito-PN exhibits a fast response for ONOO− (<5s), which is favorable for monitoring the highly reactive and short-lived ONOO− in the biological systems. The excellent sensing properties of probe Mito-PN enable its applications in the tracking of the fluctuations of endogenous ONOO− levels in the mitochondria of living cells.