The invention relates to the technical field of polypeptide preparation, in particular to cyclic hexapeptide as well as a preparation method and application thereof. The preparation method comprises the following steps: reacting fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl acyl-L-
lysine with 2-chlorotrityl
chloride, and removing the protection of trimethylsilylethoxycarbonyl, so that amino is connected to dichloro resin; the preparation method comprises the following steps: taking fluorenylmethoxycarbonyl-O-tert-butyl-L-
threonine, fluorenylmethoxycarbonyl-L-
proline, fluorenylmethoxycarbonyl-L-
alanine, fluorenylmethoxycarbonyl-
aspartic acid-4-tert-butyl ester and fluorenylmethoxycarbonyl-L-
leucine as a starting point, and then removing amino protection of terminal
amino acid and carboxyl protection of the first
amino acid; and carrying out
condensation reaction and
peptide cutting reaction to obtain the cyclic hexapeptide. The preparation method disclosed by the invention is simple in steps and simple and convenient to operate, the use of tetraphenylphosphine
palladium is avoided, and the defects of low efficiency, high cost and low yield of the traditional liquid-phase cyclization method and
solid-phase cyclization method are overcome.