The kinetics of glucose oxidase (GOD) and horseradish peroxidase (HRP) on a transparent gelatin-ionic liquid functional polymer has been investigated using a colorimetric assay of H2O2 with phenol-4-sulfonic acid and 4-aminoantipyrine. as color-generating precursors. The effect of different ionic liquids 1-ethyl-3-methyl-imidazolium ethyl sulfate ([emim][EtSO4]), 1-butyl-3-methyl-imidazolium dycianamide ([bmim][N(CN)(2)]), and 1-butyl-3-methyl-imidazolium chloride ([bmim][CI]), 1-butyl-3-methyl-imidazolium tetrafluoroborate ([bmim][BF4]), gelatin type A and water on the activity of the two free enzymes was studied. The selection of the ionic liquid [emim][EtSO4) and gelatin type A with subsequent maturation of the functional polymer at water activity of 0.76 have been found as the most suitable conditions for the entrapment of both enzymes. The activity of GOD and HRP on the gelatin-[emim][EtSO4] solid disk (6.2 x 1.5 mm of diameter and thickness) formed in the bottom of the well of ELISA plate showed to be respectively 16 times and 13 times lower than in free sodium phosphate buffer. The storage stability at 4 degrees C of the both immobilized enzymes showed that GOD can retain up to 70% of the initial activity after 2 weeks, where HRP retained 91% of its initial activity. The drop cast deposition of the transparent gelatin-[emim][EtSO4] functional polymer containing the two enzymes (GOD, HRP) and the color-generating precursors on filter paper allowed to demonstrate their applicability on colorimetric glucose detection. (C) 2011 Elsevier Ltd. All rights reserved.