Asb11 gene was reported that it was associated with activation of Notch signaling pathway. The previous results in our lab showed that the gene expressed specifically in cardiac muscle and skeletal muscle. Therefore, it was hypothesized that Asb11 gene might be a candidate gene related to heart development. In this study, CRISPR/Cas9 targeting technology was employed to knockout zebrafish Asb11 gene. Firstly, the most suitable target sites in Asb11 gene were screened out on websites. Secondly, doublestranded DNA of the Asb11 gene targeting gRNA was amplified by PCR. Thirdly, the Asb11 gene gRNA and Hcas9 mRNA were coinjected into zebrafish embryos at 1cell stage. The results of the effectivenesstest showed that bases were deleted in the exon1 of the zebrafish Asb11 gene, suggesting that the CRISPR/Cas9 system was effective for knockout of Asb11 gene. After screening in the F0, F1 and F2 generations, the lines of zebrafish with Asb11 gene knockedout were established successfully. The work laid a foundation for exploring the roles of Asb11 in zebrafish heart development.