Nitroreductase (NTR) can be overexpressed in hypoxic tumors, thus the selective and efficient detection of NTR is of great importance. To date, although a few optical methods have been reported for the detection of NTR in solution, and an effective optical probe for NTR monitoring in vivo is still lacking. Therefore, it is necessary to develop a near-infrared fluorescent detection probe for NTR. In this study, five near-infrared (NIR) cyanine dyes with fluorescence reporting structure decorated with different nitro aromatic groups, Cy7-1 - Cy7-5, have been designed and explored for possible rapid detection of NTR. Our experimental results presented that only para-nitro benzoate group modified cya-nine probe (Cy7-1) could serve as a rapid NIR fluorescence-enhanced probe for monitoring and bioimaging of NTR. The structure-function relationship has been revealed by theoretical study. The linker connecting detecting group and fluorescence reporting group and the nitro group position are key factors for the formation of hydrogen bonds and spa-tial structure match, inducing the NTR catalytic ability enhancement. The in vitro response and mechanism of the en-zyme-catalyzed reduction of Cy7-1 have been investigated through kinetic optical studies and other methods. The re-sults have indicated that an electro withdrawing group induced electron-transfer process becomes blocked when Cy7-1 is catalytically reduced to Cy7-NH2 by NTR, which is manifested in enhanced fluorescence intensity during the detec-tion process. Confocal fluorescence imaging of hypoxic A549 cells has confirmed the NTR detection ability of Cy7-1 at the cellular level. Importantly, Cy7-1 can detect tumor hypoxia in a murine hypoxic tumor model, showing a rapid and significant enhancement of its NIR fluorescence characteristics suitable for fluorescence bioimaging. This method may potentially be used for tumor hypoxia diagnosis.