This work is focused on the prebiotic synthesis by a purified immobilized β-fructofuranosidase (FFase) using a by-product molasses as a substrate. When cultivated on wheat bran, the fungus Sclerotinia sclerotiorum produces FFase with interesting transfructosylating activity. The enzyme was purified by gel filtration and anion exchange chromatography to homogeneity. It showed a specific activity of 66.06 U/mg and a molecular mass of 50 kDa. The FFase was immobilized covalently on alginate and chitosan, and the immobilization yield was 90% and 81% respectively, yet the immobilization efficiency was 52% and 93% in that order. The fixed enzymes were stable at a pH varying from 4.0 to 7.0 and at a temperature ranging from 4 to 70 °C. Yet, kinetic parameters and catalytic efficiency were determined for both immobilized and free FFases. Interestingly, chitosan cross-linked enzyme activity was maintained at 89.24% level after 50 reuses during 1 week. Continuous production of fructooligosaccharides (FOS) from beet molasses in chitosan enzyme reactor was improved. The maximum production yield obtained in 12 H was 72.2% (g FOS/g Sucrose). Thin-layer chromatography analysis showed that the major products are kestose and nystose.