Abstract: A novel enzymatic-catalyzing process of monolauroyl fructose was developed.In the process immobilized Lecitase Ultra was used as biocatalyst in anhydrous 2-methyl-2-butanol.Silica gel column chromatography was used to separate and purify the fructose ester.The qualitative analysis condition of TLC was confirmed and the purity was identified by RP-HPLC-ELSD.Structure was verified by HPLC-ESI-MS, FT-IR, 13C NMR.As a result, a mixture of the C-1 and C-6 monoacylated frutose esters, four isomers of monolauroyl fructose were obtained:l-lauroyl-β-D-fructopyranose, 1-lauroyl-α-D-fructopyranose, 1-lauroyl-β-D-fructofuranose, 6-lauroyl-β-D-fructofuranose, and l-lauroyl-β-D-fructopyranose was the highest proportion.