Hypoxia detection is emphasized with attention due to tumor and related diseases diagnosis, which could provide useful methods for exploring the mechanism of hypoxic tumor. Herein, we report two unprecedented hypoxia-sensitive probes that specifically switch-on their near-infrared fluorescence signals in the presence of hypoxia up-regulated enzymes (nitroreductase and cytochrome P450 reductase). The probes were designed by featuring the decomposition of IR-780 coupled to hypoxia activatable p-nitrobenzyl or azo moiety, which exhibit near-infrared fluorescence emission, high sensitivity, selectivity, stable photostability, and low cytotoxicity. Besides, the joint use of two probes could differentiate the 4T1 and HepG2 cells lines through fluorescence signals successfully. More importantly, applied to monitor hypoxia in 4T1 tumor-bearing BALB/c mice, the two probes have ideal biodistribution with passive accumulation and fast clearance, and there is negligible organ damage by hematoxylin and eosin staining analysis. To the best of our knowledge, there is no fluorescent probe for hypoxia detection via joint hypoxia regulated enzymes reported so far. This method may be of great potential use in cancer and other relevant diseases diagnosis.