CRISPR/Cas9 technology, developed rapidly in recent years with a high targeting efficiency, is considered a revolutionary field of genetic technology. TitinCap gene is a candidate gene for zebrafish cardiac development, and there is no knockout strain of zebra fish TitinCap gene in the world so far. In order to study the mechanism of the TitinCap gene in heart development, here the CRISPR/Cas9 gene targeting technique was used to establish the TitinCap gene knockout strain in zebrafish. The sequencing results showed that the embryos injected with CRISPR/Cas9 gRNAs had bimodals, indicating that there was a base deletion or insertion near the target site. The sequencing results demonstrated that the gRNA was effective. In the screened F0 mutants, the sequencing results also showed positive results. All these results indicated that the TitanCap gene of the zebrafish was successfully knocked out with the CRISPR/Cas9 gene targeting technique, and the zebra fish of the TitinCap gene knockout was obtained.