We used Aspergillus oryzae mycelia containing a transfructosylation-catalyzing enzyme, β-fructofuranosidase, to establish a methodology for the large-scale production of N -acetylsucrosamine in a column bioreactor. A . oryzae mycelia containing the enzyme were grown in liquid culture and immobilized on Perlite macro-porous amorphous volcanic glass beads, which were then packed into the column reactor. Passing a solution containing 10% sucrose and 20% 2-acetamido-2-deoxy-D-glucose through the reactor resulted in the steady production of N -acetylsucrosamine for at least 30 days and provided a yield of at least 27.5% (molar yield) from sucrose. N -Acetylsucrosamine was isolated easily and in high yield (from 83.4 to 96.9%) from the column reactor eluate after preprocessing by yeast β-fructofuranosidase or by whole yeast cells.