Sucrose at 10 to 20% (w/v) was the best carbon source for the production of β-fructofuranosidase by Aureobasidium sp. ATCC 20524. At higher concentrations, it arrested growth. Glucose and fructose were also good carbon sources for the enzyme production. Yeast extract at 1.5 to 2% (w/v) was the best nitrogen source for the enzyme production and for cell growth. Addition of NaNO3 (1 to 2%, w/v) and MgSO4·7H2O (0.5 to 1.5%, w/v) to the cultivation medium increased the intracellular enzymatic activity. The total enzymatic activity and cell growth reached 1.2×10(4) U/flask and 2.5 g dry cell/flask, respectively after 48 h.