d-Psicose has been drawing increasing attention in recent years because of its medical and health applications. The production of d-psicose from d-glucose requires the co-expression and synergistic action of xylose isomerase and d-psicose 3-epimerase. To co-express these genes, vector pET-28a(+)-dual containing two T7 promoters and RBS sites and an Multiple Cloning Sites was constructed using the Escherichia coli expression plasmid pET-28a(+). The xylose isomerase gene from E. coli MG1665 and the d-psicose 3-epimerase gene from Agrobacterium tumefaciens CGMCC 1.1488 were cloned and co-expressed in E. coli BL21(DE3). After 24h incubation with the dual enzyme system at 40°C, the sugar conversion ratio from d-glucose to d-psicose reached 10%. The optimal conditions were 50°C, pH 7.5 with Co2+ and Mg2+. The d-psicose yields from sugarcane bagasse and microalgae hydrolysate were 1.42 and 1.69g/L, respectively.