Abstract: Fructooligosaccharides (FOS) are fructose oligomers containing a glucose residue at the end of the chain and its production takes place by the action of fructosyltransferase in plants and micro-organisms. FOS have functional properties that make them important food ingredients, with functions of regulating lipid metabolism, increased intestinal calcium absorption and being prebiotic selectively stimulating the growth and/or activity of beneficial bacteria to the body. Bacillus subtilis natto produces levan by fermentation in medium rich in sucrose. Levan is synthesized by the enzyme levansucrase which is also responsible for the synthesis of FOS. The aim of this study was to evaluate by statistical methodology, using a central composite factorial design, the influence of varying sucrose concentration (X1), pH (X2) and agitation (X3) in the formation of FOS by B. subtilis natto CCT 7712. Fermentations were conducted in 125 mL Erlenmeyer flasks containing 25 mL of culture medium, the media were inoculated with 0.2 g L-1 cells and incubated on orbital shaker at 37°C for 24 h, the other parameters were adjusted according to the experimental design. The production of FOS was accompanied by High Performance Liquid Chromatography (HPLC) using kestose and nystose as standards. The analysis showed that the pH was significant. The optimum conditions were determined sucrose concentration of 300 g L-1, pH 7.7 and agitation 234 rpm, where production was 98.86 g L-1 and did not differ statistically from the expected value of 106.98 g L-1.