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Sensitive optical imaging of active biomolecules in the living organism requires both a molecular probe specifically responsive to the target and a high-contrast approach to remove the background interference from autofluorescence and light scatterings. Here, a responsive probe for ascorbic acid (vitamin C) has been developed by conjugating two nitroxide radicals with a long-lived luminescent europium complex. The nitroxide radical withholds the probe on its "off" state (barely luminescent), until the presence of vitamin C will switch on the probe by forming its hydroxylamine derivative. The probe showed a linear response to vitamin C concentration with a detection limit of 9.1 nM, two orders of magnitude lower than that achieved using electrochemical methods. Time-gated luminescence microscopy (TGLM) method has further enabled real-time, specific and background-free monitoring of cellular uptake or endogenous production of vitamin C, and mapping of vitamin C in living Daphnia magna. This work suggests a rational design of lanthanide complexes for background-free small animal imaging of biologically functional molecules.