This study was designed to develop a process for the immobilization of xylose isomerase(D-xylose ketol isomerase, EC 5.3.1.5) from Streptomyces griseolus previously isolated by the authors and its application on a pilot plant scale for the production of high fructose corn syrup. The biomass which has endo-excreted xylose isomerase was homogenized under a pressure of 500㎏/㎠ and 90.8% of the enzyme recovery of the native activity was obtained as compared to 54.7% recovery by the lysozyme treatment. Ionic bonding method was adopted for the enzyme immobilization due to its many reported merits. It was found that the porous resins such as Diaion HP 20, Duolite A-7, Amberlite IRA 93 and 94 were effective in immobilizing the enzyme. In addition, it was disclosed that the regeneration form of BO₄^-- is effective for Amberlite IRA 93 and HCO₃^- for Diaion HP 20. Optimal immobilization condition for Amberlite IRA 93 was pH 8.0 and 55℃ yielding 80.6% of immobilization. Activity decay test showed half life of the immobilized enzyme with Amberlite IRA 93 was more than 24 days at 65℃. The carrier was evaluated to be resuable and its result showed the relative immobilization yields were 98.2, 93.3, 90.7 and 87.5%, respectively at second, third, forth and fifth rebinding test of the enzyme on Amberlite IRA 93. Optimal temperature of the immobilized enzyme was slightly lowered and the range widened to 60∼70℃, while optimal pH moved toward 8.0∼8.3 in its isomerization reaction. The trial production result of high fructose corn syrup in pilot scale immobilization showed that one liter of immobilized xylose isomerase (350 IXIU/㎖-R) is capable producing about 293ℓ high fructose corn syrup(75% dry substance) in 30 days.