A glucose sensor was fabricated by the use of a microemulsion. The oil-in-water microemulsion was a simple three-component system consisting of a non-ionic surfactant, monomeric thiophene and aqueous buffer, without the use of an organic solvent. Enzyme was entrapped by the deposition of a thin layer of non-conducting polythiophene. This enzyme layer could be restored readily by the same microemulsion polymerization procedure when the enzyme was depleted. Application of this glucose sensor system in a flow-cell electrode was performed successfully. A linear range of over three orders of magnitude of glucose concentrations and a detection limit at the nanomole level were readily achieved. The capability of this sensor was demonstrated in connection with high-performance liquid chromatography for the direct sensing of glucose in fresh orange.