Hypoxia is one of major phenomena in metastatic and solid tumors and affects the therapy of tumor by preventing the proper metabolism of anticancer agents. Many clinical research has revealed that tumor hypoxia shows a correlation with a resistance to chemotherapy and radiotherapy. Also, it has been known that the neovascularization in growing tumors leads to the angiogenesis of diverse tumors which results in poor prognosis and resistance to chemotherapy and radiotherapy. Since the discovery of 2-nitroimidazoles as a functional group capable of making irreversible adducts with nucleophilic macromolecules in hypoxic cells, hypoxia-activated drugs have been of great interest and investigated to develop diagnostic and therapeutic agents targeting hypoxia in various types of cancers. With the recent advance in optical sensing devices, fluorescence technology has been of extensive interest in many areas including biology and medicines as well as materials due to its high sensitivity. Of various fluorophores, only a few fluorescent probes have been discovered as activity-based fluorescent probes that can detect nitroreductase (NTR) in hypoxic cells. Our group have tried to discover new rhodol-based fluorescent probes with various functional groups detecting hypoxia because rhodol fluorophore shows high fluorescence in aqueous solution in broad ranges of pH and are also known to be resistance to photobleaching. Herein, we describe new rhodol-based fluorescent probes capable of detecting and imaging NTR, highly expressed protein in hypoxic cells, and evaluation of their photochemical properties as activity-based fluorescent probes targeting hypoxia.