Flow injection analysis technique for monitoring of xylitol concentrations in biological processes has been developed using xylitol oxidase (XYO) immobilized on VA-Epoxy Biosynth carrier. The immobilized XYO cartridge has been integrated into a FIA system with an oxygen electrode and systematically investigated with regards to the factors which can affect the activity of the immobilized XYO, such as pH, temperature, salt concentration etc. The activity of the immobilized XYO increased with the temperature ($19.0 - 29.0^{circ}C$) and sample injection volume ($75-250muL$) and molarity of potassium phosphate buffer (0.1-1 M), but it reached the highest value at pH 8.5. The XYO-FIA system has been also applied for on-line monitoring of xylitol concentrations in a reactor and showed good operational stability and agreement with off-line data measured with HPLC.