Effective monitoring of essential bioindicators with high-contrast fluorescence imaging is highly crucial to reveal the pathological progression of diseases. However, most reported probes based on asymmetric amino-rhodamine (ARh) derivatives are often limited in practical application due to the low signal-to-noise ratios. Herein, a new fluorophore, 3-methoxy-amino-rhodamine (3-MeOARh), with improved fluorescence quantum yield (0.51 in EtOH) is designed and synthesized by introducing methoxy group in the ortho-position of amino in asymmetric amino-rhodamine. Notably, the good properties of the ortho-compensation effect further effectively enable the construction of an activatable probe with a high signal-to-noise ratio. As a proof of concept, the probe (3-MeOARh-NTR) was successfully synthesized for nitroreductase detection with high selectivity, excellent sensitivity, and good stability. More importantly, the relationship between drug-induced kidney hypoxia and elevated nitroreductase concentration was first uncovered in living tissues through high-contrast imaging. Therefore, the study presents the activatable probe for kidney hypoxia imaging while highlighting the 3-MeOARh structure with a satisfactory signal-to-noise ratio. It is believed that 3-MeOARh can serve as an efficient platform for activatable probe construction to reveal the pathological progression of different diseases.