BACKGROUND Fructooligosaccharides (FOS) are prebiotic sugars used in the production of functional foods. These sugars can be produced by the transfructosylation reaction of sucrose catalyzed by fructosyltransferase enzymes (FTase, E.C.2.4.1.9). The aim of this work was to evaluate the use of cells of Aspergillus oryzae IPT‐301 as biocatalysts for the sucrose transfructosylation reaction in a packed bed reactor (PBR). RESULTS The morphology of the cells was observed by scanning electron microscopy, and BET specific surface areas were examined by nitrogen physisorption The highest transfructosylation activity (660 U g−1) of the cells was obtained at 25 min of reaction by adopting a reaction temperature of 50 °C, sucrose concentration of 470 g L−1, and volumetric flow of 15 mL min−1. The rise in volumetric flow provided an increase in the transfructosylation activity, but also a faster deactivation of the biocatalyst. Enzyme kinetics was described using the Michaelis–Menten model, with a Vmax of 632.8 U g−1 and a km of 160.7 g L−1. The cells showed constant enzymatic activity for 12 h of reaction in the PBR operated with discontinuous flow at flow rates of 5 mL min−1 and 11.5 mL min−1, and in the PBR operated with continuous flow at a flow rate of 5 mL min−1. The transition from diffusional to kinetic regimes was observed starting from 11.5 mL min−1. CONCLUSION The results obtained suggest a high potential of application of the whole A. oryzae IPT‐301 cells for continuous FOS production in PBR. © 2022 Society of Chemical Industry (SCI).