Using L-proline and L-serine as raw materials, EDC·HCl as coupling reagents, L-N-acetyl-pro-N-Fmoc-L-ser-(O-allyl) dipeptide ester were obtained by coupling reaction of L-N-acetyl-proline and N-Fmoc-L-serine allyl ester. After deprotecting Fmoc, the substitution reaction of obtained compound and 2,3,4-tri-O-acetyl-6-O-triflyl-α-D-glucopyranoside occurred, giving N-Acetyl-L-pro- N-glucopyranoside-L-ser dipeptide ester. The structures of the final product and intermediates were characteried by ¹H NMR, HR-MS.