Nitroreductases (NTRs) are known to be able to metabolize nitro-substituted compounds in the presence of reduced nicotinamide adenine dinucleotide (NADH) as an electron donor. NTRs are present in a wide range of bacterial genera and, to a lesser extent, in eukaryotes hypoxic tumour cells and tumorous tissues, which makes it an appropriate biomarker for an imaging target to detect the hypoxic status of cancer cells and potential bacterial infections. To evaluate the specific activation level of NTR, great efforts have been devoted to the development of fluorescent probes to detect NTR activities using fluorogenic methods to probe its behaviour in a cellular context; however, NTR-responsive MRI contrast agents are still by far underexplored. In this study, -nitrobenzyl substituted -weighted magnetic resonance imaging (MRI) contrast agent Gd-DOTA-PNB (probe ) has been designed and explored for the possible detection of NTR. Our experimental results show that probe could serve as an MRI-enhanced contrast agent for monitoring NTR activity. The response and mechanism of the NTR catalysed reduction of probe have been investigated through LC-MS and MRI. -nitrobenzyl substituted probe was catalytically reduced by NTR to the intermediate -aminobenzyl substituted probe which then underwent a rearrangement elimination reaction to Gd-DOTA, generating the enhanced -weighted MR imaging. Further, LC-MS and MRI studies of living have confirmed the NTR activity detection ability of probe at a cellular level. This method may potentially be used for the diagnosis of bacterial infections.