Abstract A chemiluminescence fiber optic system coupled to flow injection analysis (FIA) and ion exchange chromatography has been developed for determining glucose in blood and urine. Immobilized glucose oxidase acted on β- d-glucose to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. Endogenous ascorbic acid and uric acid present in urine or blood samples were effectively retained by an upstream acetate anion exchanger. In addition, acetaminophen could also be adsorbed by this ion exchanger. The detection system exhibited a sensitivity of 1.315 ± 0.044 RU μ m -1 for glucose with a minimum detection level of 1 μ m. When applied for the determination of urinary and blood glucose levels, the results obtained compared well with those of the reference hexokinase assay. Immobilized glucose oxidase was reused for over 500 analyses without losing its original activity. A conservative estimate for the reuse of the acetate ion exchange column was about 100 analyses.