The invention discloses a CRISPR / Cas9 system for knocking out the dmrt1 gene at double gRNA sites in yellow catfish. The CRISPR / Cas9 system includes the following steps: (1) Target site 1 is designed on the dmrt1 gene of yellow catfish On the first exon, the target site 2 is designed on the third exon; (2) design primers according to the target site sequence of step (1) to detect the accuracy of the target site in the broodstock, use dmrt1 E1 F and dmrt1 E1 R amplifies target site 1 and nearby sequences, and uses dmrt1 E3 F and dmrt1 E3 R to amplify target site 2 and nearby sequences; (3) Using pUC19‑gRNA‑scaffold plasmid as a template, use dmrt1 E1 gRNA F and gRNA R was used to amplify the gRNA1 fragment by PCR, and dmrt1 E3 gRNA F and gRNA R were used to amplify the gRNA2 fragment by PCR; using the above PCR product as a template, gRNA was obtained by in vitro transcription and purification; (4) the pXT7‑hCas9 linearized plasmid was used as Template, transcribed and synthesized Cas9 mRNA in vitro; (5) Microinjected Cas9 mRNA and two gRNAs into one-cell embryos of yellow catfish; (6) Detected the mutation type and calculated the gene editing rate.