Simulated moving bed (SMB) processes have been widely used in the sugar industries with ion-exchange resin as a stationary phase. D-psicose, a rare monosaccharide known as a valuable pharmaceutical substrate, was synthesized by the enzymatic conversion from D-fructose. The SMB process was adopted to separate D-psicose from D-fructose. Before the SMB experiment, the reaction mixture including D-psicose and D-fructose was treated by a deashing process to remove contaminants, such as buffers, proteins, and other organic materials. Four columns packed with Dowex 50WX4-Ca2+ (200-400 mesh) ion-exchange resins were used in the four-zone SMB. Single-step frontal analysis was performed to estimate the isotherm parameters of each monosaccharide. The operating conditions of the SMB process were determined based on the Equilibrium Theory. According to the simulation of the SMB process, the purity and yield of extract product (D-psicose) achieved were 99.04 and 97.46%, respectively and those of raffinate product (D-fructose) were 99.06 and 99.53%, respectively. Under the optimized operating condition, complete separation (extract purity = 99.36%, raffinate purity = 99.67%) was achieved experimentally.