The invention discloses a construction method and application of mycobacteria genetic engineering bacteria, and belongs to the technical field of genetic engineering. A recombinant plasmid is transformed into a mycobacteria LY-1 cell, sgRNA can distinguish a specific region on a genome and lead Cas9 protein to be combined to a target site; and under the action of the protein, a double-strand breaknotch is formed in the target site, and most of cells are dead. Under the action of recombinase, a homologous repairing sequence led along with the recombinant plasmid is integrated to the notch through double exchange, and the cells successfully repaired homologously by genes can survive, and form genotypes which are artificially designed for gene inactivation, exogenous gene insertion and the like. The knockout efficiency for the same gene by the constructed CRISPR-Cas9 system is 60%, and compared with a conventional mycobacteria gene editing manner, the constructed CRISPR-Cas9 system is more convenient and higher in efficacy.