Peroxynitrite (ONOO-), a highly reactive nitrogen species (RNS) generated mainly in mitochondria, has been identified to be associated with numerous pathophysiological processes, and thus accurate ONOO- imaging with superior sensitivity and selectivity is highly desirable. Herein, we prepared a new type of carbon quantum dots (CQDs) with mitochondria-targeting function without the aid of any targeting molecules via a simple one-step hydrothermal route. The as-prepared CQDs not only displayed relatively uniform size distribution, few surface defects, high photostability, and excellent biocompatibility but also exhibited good selective fluorescence turn-off response toward ONOO-, owing to the oxidation of amino groups on the surface of carbon dots. A great linear correlation between the quenching efficiency and ONOO- concentration in the range from 0.15 to 1.0 μM with a detection limit of 38.9 nM is shown. Moreover, the as-prepared CQDs acting as a functional optical probe through a self-targeting mechanism were successfully applied for in situ visualization of endogenous ONOO- generated in the mitochondria of live cells, providing great promise for elucidating the complex biological roles of ONOO- in related pathological processes.