Poly(vinyl alcohol)-pyrene-glucose oxidase (PVA-Py-GOx), a water-soluble polymer possessing both fluorescent and oxidant sites in the structure, is synthesized by "click" chemistry and modification processes and characterized. The morphology of PVA-Py-GOx was characterized with atomic force microscopy (AFM), and a heterogeneous morphology due to the incorporation of GOx was observed. The capability of PVA-Py-GOx to act as a bioprobe for fluorescence sensing of glucose is examined. The postulated fluorescence mechanism for glucose analysis is based on the consumption of glucose by dissolved oxygen and GOx present in the structure. Thus, the fluorescence intensity of pyrene groups of the probe increases by the elimination of fluorescence quenching by oxygen. Glucose concentration was analyzed quantitatively from 0.25 to 3.0 mM by the fluorescence measurement. The effect of pH and amount of PVA-Py-GOx was also studied. The proposed system was applied to analyze glucose in real samples and compared with those obtained from commercial kits.