A coupled enzyme system for the detection of D-xylose and D-xylulose is presented. The system is based on three consecutive enzymatic steps. The enzymes xylose isomerase (XI), mutarotase (MT), and glucose dehydrogenase (GDH) are coimmobilized on controlled pore glass and packed in a bed reactor. The relative amount of enzymes, ie., enzyme ratio, plays a critical role in driving the overall reaction, resulting in a system with linear response characteristics and an operational range of several orders of magnitude. Three different enzyme ratios are assayed to achieve maximum conversion efficiencies for xylose and xylulose. The highest enzyme unit ratio assayed 13.4 of GDH to XI, gave the highest apparent pseudo-first-order rate constant showing the importance of tile last enzymatic reaction in the coupled system to make the overall reaction thermodynamically favorable. A pH of 7.0 was found to be an optimum compromise for the multienzyme system. Sensitivity was dependent on NAD(+) concentration. The study was carried out in a flow injection system. The optimized reactor has been applied for the catalytic detection of pentoses in flow injection analysis (FIA) and liquid chromatography (LC).