Abstract: Rare sugars D- sorbose and D- psicose were simultaneously synthesized with L- rhamnulose1-phosphate aldolase ( RhaD) at low cost. To avoid direct use of dihydroxyacetone phosphate ( DHAP) , a rather expensive and unstable substrate, a one- pot four- enzyme strategy based on RhaD and phosphatase ( AP or YqaB) was adopted to optimize the synthesis of D- sorbose and D- psicose with a cheap substrate DL- glycerol3-phosphate as the starting material.The products were firstly detected by TLC and HPLC, then purified by silica gel、P-2 gel filtration and Ca2 +exchange resin.Lastly, the products after purification were identified with1H NMR.The results demonstrated that one-pot four-enzyme strategy and purification method could be efficiently applied for the practical production of D- sorbose and D- psicose. This approach could provide the theory basis for the synthesis of more other rare sugars and their derivatives.