We describe a new approach to glucose sensing using polarization measurements in the presence of a stretch-oriented reference film. The method relies on measurement of the polarized emission from the reference film and of a fluorophore which changes intensity in response to glucose. A glucose-sensitive fluorescent signal was provided by the glucose/galactose binding protein from E. coli. This protein was labeled with an environmentally sensitive fluorophore at a single genetically inserted cysteine residue, and displayed decreased fluorescence upon glucose binding. Using the protein and the reference film we observed glucose-sensitive polarization values for micromolar glucose concentrations. This method of polarization-based sensing is generic and can be used for any sensing fluorophore which displays a change in intensity. In principle, one can construct simple and economical devices for this type of glucose measurement. © 1999 Society of Photo-Optical Instrumentation Engineers.