The use of PSU-Py prepared by click chemistry as a platform in membrane-bottom microwell plates for oxidase and hydrolase/oxidase-based enzyme assays is studied. For the GOx assay, the postulated fluorescence mechanism is based on the consumption of glucose by dissolved oxygen and GOx in the microwell plates covered with the PSU-Py membrane. For the AG-GOx assay, maltose is used as AG substrate and hydrolyzed to glucose which is then oxidized by the GOx activity. It is shown that the PSU-Py membrane acts as a fluorescence indicator of the enzymatic reactions, and both GOx and AG/GOx enzyme assays are successfully applied for glucose, maltose and acorbose analysis in the range 0.125-2.0 x 10(-3) M glucose, 0.05-0.5 x 10(-3) M maltose, and 0.0125-0.1 mg . mL(-1) acorbose, respectively.