Abstract: The invention provides a self-cleaving protein and a protein preparation method by employing a self-cleaving protein. The self-cleaving protein comprises an intein domain, wherein the intein domain contains at least one residue mutation at its N-terminus. The self-cleavage is prohibited under an “prohibition condition” and when the self-cleaving protein is under an “inducement condition”, the C-terminal cleavage will occur but N-terminal cleavage will not. In addition, the invention provides a fusion protein comprising the self-cleaving protein and target protein.