MicroRNA (miRNA), a type of endogenous non-coding RNA with a length of about 19~23 nucleotides, participates in post-transcriptional regulation of gene expression by affecting the stability and translation of mRNA. Bioinformatics analysis showed that the gene is highly conserved in various species. In order to study the role of miR-196a-1 gene in intestinal development, we used cloning free CRISPR/Cas9 gene editing technology to establish the zebrafish miR-196a-1 knockout lines. Firstly, two knockout sites of zebrafish miR-196a-1 gene were screened by online analysis, the two knockout sites were 132 bp apart. Next, the template guide DNA of miR-196a-1 gene was amplified by PCR. Then, the template DNA was transcribed into the sgRNA of miR-196a-1 gene. Finally, the sgRNA of miR-196a-1 gene and Cas9 protein were co-injected into zebrafish embryos at the 1-cell stage. The effectiveness of gene editing was tested after 36 hpf.The results showed that there was a deletion of 102 bp base in miR-196a-1 gene, revealing that the CRISPR/Cas9 system is effective in knocking out miR-196a-1 gene. The miR-196a-1 knockout line of zebrafish was established successfully after screening the F0, F1, and F2 generations. These findings laid a foundation for exploring the role of miR-196a-1 in zebrafish intestinal development.