The invention discloses a [<18>F] AlF marked positron emission tomography (PET) polypeptide probe and a preparation method thereof. The probe comprises TMTP1 polypeptide and NOTA, which are connected together, and the structural formulae of the TMTP1 polypeptide and the NOTA are shown in the description. According to the [<18>F] AlF marked positron emission tomography (PET) polypeptide probe and the preparation method thereof, a glycine is added to the TMTP1, and a G-TMTP1 polypeptide is designed, so that the polypeptide does not influence the biological activity after being radioactively marked; furthermore, 18F is taken as a radionuclide, and the G-TMTP1 polypeptide is marked by utilizing a [<18>F] AlF-NOTA method, so that the obtained [<18>F] AlF-marked PET polypeptide probe has excellent pharmacokinetics properties, the background cleaning rate is high, the liver uptake rate is low, in-vivo stability is good, the uptake rate of tumor site is high, highly metastatic tumor can be diagnosed specifically, and the polypeptide probe can be applied to diagnosis or therapeutic evaluation of highly metastatic malignant tumor.