BACKGROUND Fructooligosaccharides (FOS) are sweeteners produced through the use of enzymes called fructosyltransferases (FTases) (EC 2.4.1.9). However, FTase reuse is meagre and the cost of the enzyme production is high, making FOS production economically unfeasible. In order to address these issues, this study aims to immobilize cells from Aspergillus oryzae IPT-301, containing FTase enzyme previously glutaraldehyde-crosslinked, by means of entrapment in calcium alginate beads. The conditions to obtain the particles, such as alginate type, mycelium mechanical dispersion, degree of crosslinking, size of the beads, and their reflections on transfructosylation activity (A(T)) were evaluated previously. RESULTS The highest A(T) used low-viscosity alginate solution (4.0% w/w) crosslinked with calcium chloride (0.2 mol L-1) and beads of mean diameter 2.15 mm. The entrapment conditions were complemented by conducting the reaction under different temperatures, pH values, substrate concentrations and by reusing the beads. The highest transfructosylation activities were obtained under reaction conditions of 50 degrees C, pH 5.5 and substrate concentration 400 g L-1. CONCLUSION The A(T) levels found with the glutaraldehyde-crosslinked and entrapped cells in calcium alginate were approximate to 70% higher than the ones obtained with free cells, not glutaraldehyde-crosslinked. Moreover, the immobilized cells (crosslinked+entrapped) retained their A(T) up to the third cycle of total sucrose depletion in the reaction medium, attesting to the potential use of the beads in continuous FOS production.