The invention provides a TEV protease mutant, a gene, a biomaterial, a preparation method, a reagent, or a reagent kit and application, and relates to the technical field of genetic engineering. Compared with a protease mutant having an amino acid sequence shown as SEQ ID NO.1, the TEV protease mutant is mutated into at least seven amino acid residues, for example 17th site is mutated into S fromT, the 56th site is mutated into V from L, the 68th site is mutated into D from N, the 77th site is mutated into V from I, the 135th site is mutated into G from S, the 219th site is mutated into V from S, and the 233th site is mutated into H from Q. The TEV protease mutant has favorable enzymatic activity, stability and specificity. The gene, the biomaterial and the preparation method of the TEV protease mutant, or the reagent or the reagent kit containing the TEV protease mutant, the application of the technical scheme, and the TEV protease mutant are all based on the same invention conception.