Fructooligosaccharides are prebiotics with numerous health benefits within which the improvement of gut microbiota balance can be highlighted, playing a key role in individual health. In this study, an integrated one-stage method for FOS production via sucrose fermentation by Aureobasidium pullulans was developed and optimized using experimental design tools. Optimization of temperature and agitation speed for maximizing the FOS production was performed using response surface methodology. Temperature was found to be the most significant parameter. The optimum fermentation conditions were found to be 32°C and 385rpm. Under these conditions, the model predicted a total FOS production yield of 64.7g_FOS/g_sucrose. The model was validated at optimal conditions in order to check its adequacy and accuracy and an experimental yield of 64.1 (±0.0)g_FOS/g_sucrose was obtained. A significant improvement of the total FOS production yields by A. pullulans using a one-stage process was obtained.