The invention discloses a method for preparation of N-[(S)-(2,3,4,5,6-pentafluorophenyloxy)phenoxy phosphoryl]-L-alanine isopropyl ester; the method comprises the steps: first of all, carrying out a reflux reaction of dichloro phenyl phosphate and pentafluorophenol for 5-15 hours in an organic solvent and under an alkaline condition; followed by, cooling the reaction liquid down to room temperature, adding L-alanine isopropyl ester hydrochloride, and carrying out a stirring reaction for 2-8 hours at room temperature; and then carrying out suction filtration, concentrating the filtrate under reduced pressure, and carrying out recrystallization treatment on the concentrated residue. The method can effectively solve the defect problems that a conventional method has harsh reaction conditions, high requirements on equipment, relatively low yield and the like, prepares the high-purity N-[(S)-(2,3,4,5,6-pentafluorophenyloxy)phenoxy phosphoryl]-L-alanine isopropyl ester with simple operation, mild conditions and relatively high yield, and has an important value on achieving scale production of the compound.