The invention discloses a method for knocking out MSTN (myostatin) genes in a targeted manner by utilizing CRISPR-Cas9. According to the method, multiple species are subjected to homology comparison, two pairs of MSTN gene target sequences are selected, two pairs of DNA double strands which have the sequences different from the target sequences, the same expression protein and the same BsmBi sticky ends are synthesized according to the design principle of PAM of gRNA, the double strands are connected with pPDNA330 plasmids, a recombinant vector carrying MSTN homologous genes of multiple species is obtained and is used for detecting the gene knockout efficiency with a fluorescent protein expression method, carrier-transfected receptor cells, having higher knockout efficiency, of multiple species are selected, gene knockout and verification are performed, and simple, efficient and accurate gene knockout on the MSTN genes of the multiple species is completed.