AbstractDue to the health-promoting effects and functional properties of inulin-type fructooligosaccharides (I-FOS), the global market for I-FOS is constantly growing. Hence, there is a continuing demand for new, efficient biotechnological approaches for I-FOS production. In this work, crude inulosucrase InuGB-V3 from Lactobacillus gasseri DSM 20604 was used to synthesize I-FOS from sucrose. Supplementation with 1 mM CaCl2, a pH of 3.5–5.5, and an incubation temperature of 40 °C were found to be optimal production parameters at which crude inulosucrase showed high conversion rates, low sucrose hydrolysis, and excellent stability over 4 days. The optimal process conditions were employed in cell-free bioconversion reactions. By elevating the substrate concentration from 570 to 800 g L−1, the I-FOS concentration and the synthesis of products with a low degree of polymerization (DP) could be increased, while sucrose hydrolysis was decreased. Bioconversion of 800 g L−1 sucrose for 20 h resulted in an I-FOS-rich syrup with an I-FOS concentration of 401 ± 7 g L−1 and an I-FOS purity of 53 ± 1% [w/w]. I-FOS with a DP of 3–11 were synthesized, with 1,1-kestotetraose (DP4) being the predominant transfructosylation product. The high-calorie sugars glucose, sucrose, and fructose were removed from the generated I-FOS-rich syrup using activated charcoal. Thus, 81 ± 5% of the initially applied I-FOS were recovered with a purity of 89 ± 1%.