In this paper a detailed mathematical model is presented for the fermentative production of fructo-oligosaccharides with Aspergillus sp. The model accounts for hydrolysis and transfructolization reactions, as well as biomass formation and it contains 27 parameters that were determined from experimental data using a System Biology toolbox with the Simulated Annealing method for curve fitting. Several additional experiments were performed in bioreactors where the time variation of 7 state variables (Sucrose, Glucose, Fructose, 1-Kestose, Nystose, 1-fructosyl nystose and Biomass) was measured. Experimental data were compared with results from simulations using the estimated parameters and it was verified that the model can predict the FOS production profile. The good agreement between simulated and experimental data was verified by calculating the relative percentage deviation modulus, which was lower than 10% for all cases except one. The derived and validated model can be used for process optimization, for example for indicating which fed-batch strategy could be used to improve the production of FOS while minimizing glucose concentration.