Hypochlorite anion (ClO-) has been recognized as host defense destructing incursive bacteria and pathogens, a signal molecule inducing occurrence of apoptosis and a noxious agent when it is overproduced. It is significant to detect ClO- in mitochondria for getting meaningful physiological and pathological information. Compared with the fluorescence probes of emission wavelength in ultraviolet or visible region, those with near-infrared (NIR) fluorescence signal are advantageous due to the deeper tissue penetrability and less photo-bleaching effect. In this work, a new "off-on" NIR ClO--specific fluorescence probe (Mito-NClO) especially located in mitochondria was designed and synthesized by condensation of diaminomaleonitrile with a new fluorophore (Mito-NCHO). A marked "turn-on" NIR fluorescence signal was observed on account of the oxidation of the imine bond by NaClO. Moreover, in the range from 0 to 20 μM, this probe had the capability to quantitatively detect ClO- with a detection limit as low as 90.2 nM. Additionally, the probe exerted other excellent properties, including larger stokes shift (117 nm), better aqueous solubility, high selectivity toward ClO-, rapid response and selective mitochondrial location. Furthermore, the bio-imaging experiments clearly demonstrated that Mito-NClO facilitated the visualization of exogenous and endogenous ClO- in living HeLa cells and zebrafish model. Therefore, we speculate that the probe Mito-NClO can be served as an ideal tool for the monitoring of ClO- in biosystems.