Nitroreductase (NTR) is to be pivotal in the biodegradation of nitroaromatics. NTR is produced in tumor tissues under hypoxic conditions, which is one of the markers for early tumor diagnosis. In this study, a novel probe FD-NTR was developed for NTR detection. Probe FD-NTR can exhibit a specific reaction with NTR in the presence of NADH. The probe displayed satisfactory selectivity and sensitivity towards NTR with a calculated detection limit of 12 ng/mL. Under the conditions of low cytotoxic hypoxia, the FD-NTR probe has shown successful application in imaging both MCF-7 cells and tumor tissues, which indicated that the FD-NTR probe holds promising application prospects for detecting NTR in tumors.