Cells of Zymomonas mobilis possessing glucose-fructose oxidoreductase and gluconolactonase were permeabilized with toluene and co-immobilized with invertase within a gelatin membrane. This membrane was coated over a pH electrode, and the resulting biosensor was placed in a flow-through cell to develop a flow injection analysis (FIA) system for the specific determination of glucose, fructose, and sucrose. Peak height resulting from the production of hydrogen ion was correlated with sugar concentration, and the effect of operating variables on the response characteristics of the FIA system was investigated on the basis of theoretical and experimental analyses. Under the optimized conditions, the calibration curves for glucose, fructose, and sucrose were linear up to 8, 80, and 60 g/L, respectively. The FIA system was applied to the online monitoring of glucose production in the enzymatic hydrolysis of cellulose, and the glucose concentrations determined using the FIA system coincided well with those determined by the conventional enzymatic method.