The invention relates to a rape multiple ALS (acetolactate synthase) inhibitor type herbicide-resistant gene based on in-vitro site-specific mutagenesis and application, and belongs to the technical field of biology. The gene mALS3 is obtained by introducing a new mutant site into the ALS3 gene sequence of the herbicide-resistant rape M342, and the site is positioned at the 560th site of the ALS3 gene; the original gene is alanine with a C alkaline group at the 187th site, the C alkaline group is mutated into a T alkaline group, and valine is formed at the 187th site. The rape mALS3 gene with double mutation sites A187V and W556L is obtained through an in-vitro site-specific mutation technique. The double mutation sites are not reported in the literatures of the rape genome. After preliminary functional verification of transgenic arabidopsis, the rape mALS3 gene with double mutation sites has the advantages that the obvious resistance on multiple ALS inhibitor herbicides, such as imidazolamide, tribenuron-methyl, difluorosulfonamide and bispyribac-sodium, is realized; the resistance gene can be used for the culturing of herbicide-resistant crops.