The invention discloses a high stable type MazF mutant and application thereof. The present invention provides a protein as shown in 1) or 2) as follows: the protein as shown in the 1) is a protein obtained by replacement of forty-eighth site cysteine in a wild-type MazF protein amino acid sequence with alanine, phenylalanine, leucine or isoleucine; the protein as shown in the 2) is a protein derived by addition of a tag sequence at the terminal of the amino acid sequence of the protein as shown in the 1). Experiments prove that the amino acid of the forty-eighth site of the wild-type MazF protein amino acid sequence is replaced, and under the condition of maintaining endonuclease activity equivalent to that of a wild-type MazF toxic protein, the mutant effectively improves activity retention time and structural stability. The mutant can be used in all types of synthetic biology modules established on the basis of a mazEF toxin-antitoxin system, and can be widely used in the study of the mechanism of bacterial TAs suicide, antibacterial and antiviral study.