Peroxynitrite (ONOO−), as a strong oxidative biomolecule, is considered a potential biomarker of drug-induced liver injury (DILI) that could disturb biochemistry processes and cause a great variety of liver diseases. However, it remains a significant challenge to discover quickly and track accurately ONOO− in vivo environments even if these active substances have aggravated liver damage. Herein, we developed a peroxynitrite-activable mitochondria-targeted phosphorescent chemodosimeter (Ru-Mit) for DILI monitoring, which was designed by covalently linking ruthenium(II) complex with the strong electron-withdrawing malononitrile moiety. In the presence of ONOO−, the chemodosimeter hard to be oxidized exhibited excellent turn-on phosphorescent response at 610 nm with a longer lifetime (∼ 300 ns). The sensing mechanism indicated the malononitrile group in Ru-Mit was removed via the hydrolysis of C=C bond, accompanied by the change of electronic structure and the enhancement of radiative transitions. Further, reliable signals are capable of not only imaging exogenous and endogenous ONOO− in mitochondria, but also visualizing APAP-induced and tolcapone-induced ONOO− generation in HepG2 cells by confocal luminescence imaging. This work might provide a new molecular platform to design and develop ONOO− probes, and it might be used as a practical tool to detect ONOO− for the pathogenesis research of DILI.