The invention provides modification and application of nitrile hydratase amino acid motif, and belongs to the technical field of bioengineering. According to the invention, nitrile hydratase derived from pseudonocardia thermophila is modified, the NO.46 site on a beta subunit and the NO.6 site and NO.126 site on an alpha subunit of a wild type are mutated respectively, and the specific enzyme activity of a constructed mutant is remarkably improved and can be improved by 1-5 times compared with that of the wild type. Moreover, the substrate spectrum is also broadened, the mutant has a very goodcatalytic effect on isobutyronitrile, n-valeronitrile, acrylonitrile, nicotinonitrile, 2-cyanopyrazine, benzonitrile, cinnamonitrile, naphthonitrile and the like, and the catalytic performance on corresponding substrates is also remarkably improved. Therefore, the obtained nitrile hydratase mutant can be suitable for more production scenes, and the production efficiency is improved.