Disclosed is a process for the regioselective preparation of glycerol derivative in a high efficiency and yield. The process for the regioselective preparation of 1-R1-2-R2-3-acetyl-glycerol derivative comprises the steps of: obtaining 1-R1-3-protecting group-glycerol by introducing a protecting group to 3-position of 1-R1-glycerol; obtaining 1-R1-2-R2-3-protecting group-glycerol by introducing R2 group into 2-position of 1-R1-3-protecting group-glycerol; and carrying out the deprotection reaction and the acetylation reaction of 1-R1-2-R2-3-protecting group-glycerol at the same time. Wherein, R1 and R2 are fatty acid groups having 16 to 22 carbon atoms, and are different from each other; and the protecting group is trityl group or trialkylsilyl group.