Rare sugars D-psicose shows great potentials in many field, such as food, pharmaceutical and medicinal industries. However, large scale synthesis of D-psicose with a high yield still remains a challenge. In our previous study, DHAP-dependent aldolase L-rhamnulose-1-phosphate aldolase (RhaD) from Escherichia coli lost its stereoselectivity when accepting D-glyceraldehyde as the acceptor. As a result, two rare sugars D-psicose and D-sorbose were generated with a ratio of 1∶1. In order to optimize the stereoselectivity of RhaD to exclusively produce D-psicose, site-saturation mutagenesis of tyrosine 152 of RhaD with other nineteen amino acids were performed. The results showed that D-psicose was favored by RhaD Y152A mutant and the ratio was increased eight folds. This study will provide theoretical foundation for further molecular modification of RhaD for the synthesis of D-psicose as a single product.