Hypoxia　is　one　of　the　most　important　features　of　various　diseases　including　solid　tumors.　It　is　of　significant　clinical　importance　for　hypoxia　analysis　in　tumors,　and　the　analysis　of　hypoxia　can　be　moved　away　to　the　indirect　detection　of　nitroreductase.　Various　fluorescence　methods　have　been　developed　for　the　analysis　of　nitroreductase　and　tumor　hypoxia　due　to　their　simplicity,　non-invasiveness　and　excellent　spatial-temporal　resolution.　Here,　we　synthesized　a　multifunctional　fluorophore,　which　possesses　larger　π-conjugation　structure,　leading　to　the　red-shift　of　absorption　and　fluorescence　emission　to　long-wavelength　region.　This　fluorophore　(RCOH)　can　be　further　used　to　prepare　the　off-on　hypoxia　probe　(RCO-NTR)　by　introducing　the　electron　withdrawing　group　of　nitrobenzyl,　leading　to　the　quenching　of　fluorescence　emission　through　an　electron　transfer　process.　After　reaction　with　NTR　accompanied　by　NADH　as　an　electron　donor,　the　nitro　group　in　the　RCO-NTR　probe　can　be　reduced　and　then　the　RCOH　fluorophore　can　be　released　through　rearrangement　and　elimination,　leading　to　the　enhancement　of　fluorescence　signal.　Thus,　the　prepared　RCO-NTR　probe　can　be　used　for　nitroreductase　analysis,　which　can　be　further　used　for　hypoxia　imaging　at　both　cellular　and　animal　levels.　©　2020