D-Psicose can be converted from D-fructose catalyzed by D-psicose-3-epimerase. To realize the heterologous expression of D-psicose-3-epimerase (DPEase), the primers were designed to clone and isolate the gene. The DNA sequences and structural characteristics of DPEase were analyzed by bioinformatics. The results showed the gene possessed 870bp open reading frame and encoded 289 amino acids. Alpha helix, beta folding, and random coil respectively accounted for 38.41%, 47.06%,and 14.53% in the secondary structure of the protein. The protein located at the cell membrane belonged to a hydrophilic protein without the signal peptide and transmembrane region. The prokaryotic expression vector was constructed and transferred into the E. coli BL21. The molecular weight of DPEase was appropriately 33kDa. After an induction of 1mmol/L IPTG for 28h, the protein amount and enzymatic activity of the recombinant E. coli BL21 were 0.32g/L and 3.8U/mL. The DPEase gene of A. tumefaciens could be expressed in the engineered E. coli.