The operational conditions for xylitol production by fermentation of sugarcane bagasse hydrolysate in a fluidized bed reactor with cells immobilized on zeolite were evaluated. Fermentations were carried out under different conditions of air flowrate (0.0125–0.0375 vvm), zeolite mass (100–200 g), initial pH (4–6), and xylose concentration (40–60 g/L), according to a 24 full factorial design. The air flowrate increase resulted in a metabolic deviation from product to biomass formation. On the other hand, the pH increase favored both the xylitol yield (YP/S) and volumetric productivity (QP), and the xylose concentration increase positively influenced the xylitol concentration. The best operational conditions evaluated were based on the use of an air flowrate of 0.0125 vvm, 100 g of zeolite, pH 6, and xylose concentration of 60 g/L. Under these conditions, 38.5 g/L of xylitol were obtained, with a YP/S of 0.72 g/g, QP of 0.32 g/L·h, and cell retention of 25.9%.