The invention provides a method for terminating biallelic gene transcription. The method comprises the steps of 1, building a SA-T2A-antibiotic screening gene-Poly DNA fragment respectively containing two different antibiotic screening genes; 2, designing gRNA according to different targeting genes, and building a Cas9/gRNA expression vector; 3, inserting the SA-T2A-antibiotic screening gene-Poly DNA fragment obtained in step 1 into the targeting genes, and building homologous recombinant vectors containing the targeting genes; 4, transferring into cells: transfecting the cells with both a pair of homologous recombinant vectors containing the targeting genes obtained in step 3 and the Cas9/gRNA expression vector containing the targeting genes obtained in step 2; 5, performing screening on the relevant antibiotic of the antibiotic genes adopted in step 1 for at least two weeks to obtain biallelic gene knock-out polyclonal cell lines. The method for terminating biallelic gene transcription can achieve the purpose that when performing inserting into an efficient fixed point, biallelic gene simultaneously knock-out cell lines can be quickly obtained.