The invention discloses a method for directional knockdown of a multi-copy gene in zebrafish genome. The method comprises the following steps: I, preparing a zebrafish embryo; II, constructing an expression vector of dCas9-Eve fusion protein; III, conducting in-vitro transcription of sgRNA, dCas9-Eve, Cas9 and gene, and purifying the sgRNA, dCas9-Eve, Cas9mRNA and gene mRNA, which are synthesized through the in-vitro transcription, by virtue of RNeasy Mini Kit; IV, identifying activity of the sgRNA; and V, knocking out expression of the multi-copy gene znfl1s by virtue of a dCas9-Eve method, and judging the situation that the gene expression is knocked out by detecting the expression of the gene by virtue of an embryo whole-mount in situ hybridization method. The method provided by the invention, by virtue of the dCas9-Eve, can specifically knock down the transcription of the gene under the circumstance of implementing the effective targeted identification of sgRNA existence of a gene promoter, so that the researched gene is prevented from being affected by MO toxicity or non-specific phenotype caused by target missing; and moreover, since an Eve inhibiting transcription structural domain is derived from zebrafish, the dCas9-Eve is applicable to knockdown of any zebrafish genes at a transcription level.