Abstract The effects of production parameters on the biotransformation of sucrose were investigated to enhance the yield of neo-FOS by Xanthophyllomyce dendrorhous cells. Cells showed optimal β-fructofuranosidase activity at neutral pH condition and the yield of neo-FOS showed no significant differences between buffer and buffer-free systems. Cell concentration negatively affected the maximum neo-FOS yield. Sucrose concentration positively increased the maximum yield of neo-FOS. Elevating the reaction temperature to 30 °C, the neo-FOS productivity increased 1.85-fold compared with that at 20 °C. Meanwhile, cell age of 32 h enabled the biotransformation of sucrose more efficiently. In addition, free cells exhibited a higher productivity over immobilized cells. The maximum neo-FOS concentration finally reached 227.72 g/l from 400 g/l sucrose under the optimal conditions.