Fructooligosaccharides (FOS) are a group of carbohydrates that have fructose units, with a glucose molecule at the end. These are structures that can be found in a wide variety of vegetables, being also produced chemically by the enzymatic transfructosylation reaction of sucrose. The present work aimed at the immobilization of the extracellular FTase enzyme from Aspergillus oryzae-IPT-301 on Eucalyptus biochar and Eucalyptus biochar functionalized with glutaraldehyde, for the enzymatic conversion of sucrose into FOS. Immobilization and reuse tests were carried out and immobilization yield and recovered activity calculated. The immobilization yield (IY) and recovered activity (RA) of the FTase immobilized on biochar were, 10.92% and 77.67%, respectively. The IY and RA of the FTase immobilized on the functionalized biochar were, 22.44% and 96.51%, respectively. The enzyme immobilized on functionalized biochar demonstrated transfructosylation activity for more cycles than the enzyme immobilized on biochar. The use of glutaraldehyde allowed 48% of initial enzymatic activity in the third cycle. Analyzes were conducted up to the sixth cycle, however, only 7% of initial enzymatic activity was achieved in the final cycle. These results suggest that functionalized biochar stands out as a support material for FTase immobilization aiming at FOS production catalyzed on heterogeneous biocatalysts.