The invention discloses an Exendin-4 derivant with Exendin-4 activity and preparation and application thereof, belonging to the technical field of biological engineering. The molecular structural formula of the derivant is any one of the following structures: Exendin-4(1-39)-Xaa40(Seq ID No.2), Exendin-4(1-39)-Xaa40-Xaa41(Seq ID No.3), Exendin-4 (1-39)-Xaa40-Xaa41-Xaa42(Seq ID No.4), Exendin-4(1-30)-Xaa31(Seq ID No.6), Exendin-4(1-30)-Xaa31-Xaa32(Seq ID No.7) and/or Exendin-4 (1-30)-Xaa31-Xaa32-Xaa33(Seq ID No.8); wherein, Xaa40, Xaa41, Xaa42, Xaa31, Xaa32 and Xaa33 are respectively any amino acid of Cys, Ala, Gly, His, Ser and Thr. The Exendin-4 derivant has the advantage that the derivant has longer in-vivo drug effect time than Exendin-4. Solid phase chemosynthesis is adopted to prepare the Exendin-4 derivant, so that production steps are few and production cost is low. The Exendin-4 derivant is particularly suitable for preparing the active ingredient of drugs for curing diabetes.