β-Fructofuranosidase production by Aspergillus oryzae IPT301 was maximized in shake flasks. Response Surface Methodology (RSM) involving Small Central Composite Design was adopted to evaluate the fructosyltransferase (FTase) activity by changing three medium component concentrations: sucrose, urea and yeast extract. The optimal set of conditions for maximum fructosyltransferase production was as follows: sucrose 320.5 g/L, urea 7.13 g/L and yeast extract 2.11 g/L. In this optimal condition, the following improvements were achieved: an increase of 48.8% in cell growth, 112% and 62% in micelial and free FTase activities, respectively, 62.8% in the ratio of fructosyltransferase/hydrolytic activities for enzyme linked to mycelium and 67.5% for free enzyme.