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the present invention provides a preparation method of Semaglutide, comprising the steps of: the fully-protected fragment of Fmoc-PEG-PEG- -Glu-octadecanedioic acid used in the fragmental synthesis of the Lys 26 side chain in the preparation method is prepared by successively coupling a chloride resin to Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic Acid mono-tert-butyl ester, cleaving, and recrystallizing to obtain PEG-PEG- -Glu-Octadecanedioic Acid mono-tert-butyl ester