Abstract: This study aim to investigate the separation and purification of neokestoe produced by sucrose biotransformation for producing neokestose. First,Pichia pastoris was used to remove the fructose and glucose of the syrup and the effects of cell concentration of Pichia pastoris and fermentation time on the purity of neokestose were investigated. Then, the neokestoe syrup was further purified using Bio- Gel P- 2 and the effects of sampling amount and flow rate on the purity and recovery of neokestose were researched. The results showed that the purity of neokestose increased from 10.92% to 19.39% when the cell concentration and reaction time were 60 g/L and 10 h,respectively. Under the optimized conditions of a flow rate of 0.1 m L/min and a sampling amount of 0.5 m L,the purity of neokestose improved from 19.39% to 76.68%. This method could be used for the preparation of neokestose.