The production of fructooligosaccharides (FOSs) from sucrose catalyzed by beta -D-fructofuranosidase was achieved with the use of immobilized mycelia of Aspergillus japonicus in gluten. When I g mycelia-immobilized particles having a cell content of 20% (w/w) were incubated with 100 ml of sucrose solution with an initial concentration of 400 g/liter, the total produced FOSs were determined to be about 61%, w/w of total sugars in the mixture after a batch reaction for 5 h. The reaction velocity increased with the cell content in the gluten matrix and reached the maximum value when the cell content was as high as 20% (w/w). As the mycelia-immobilized gluten particles were packed in to a column reactor for continuous production of FOSs, a productivity of 173 g per hour per liter of reactor volume was achieved at a flow-rate of 0.8 ml/min. The mass fraction of FOSs increased from 0.20 to 0.54 w/w as the flow-rate decreased from 1 to 0.1 ml/min, corresponding to the residence time increasing from 0.35 to 3.5 h. The mycelia were well entrapped in the gluten matrix and the cell-immobilized preparations were stable in long-term operation. Gluten was found to be adequate for using as the base material for immobilization mycelia-associated enzymes.