MYO7A is the causative gene of human Usher syndrome (US). Usher syndrome cases caused by MYO7A mutation account for 29% to 55% of Usher syndrome type 1 cases. It has been reported that MYO7A mutations can cause Usher syndrome type 1B (USH1B), including sensorineural hearing impairment and age-dependent retinitis pigmentosa (RP), whereas the molecular mechanism is still unclear. In order to elucidate the molecular mechanism of this gene in inner ear and retina development, we used cloning free CRISPR/Cas9 gene editing technology to establish the zebrafish myo7ab knockout lines. Firstly, two knockout sites of the gene were screened by bioinformatics analysis. Next, the template guide DNA of this gene were amplified by polymerase chain reaction (PCR). Then, the template guide DNA were transcribed into the guide RNA. Finally, the guide RNA and Cas9 protein were co-injected into the 1-cell stage of zebrafish embryos. After the effectiveness analysis, it was proved that the CRISPR/Cas9 is effective in knocking out myo7ab gene. The myo7ab knockout line of zebrafish was established successfully after screening. The establishment of this line laid a foundation for studying the role of myo7ab in the development of the inner ear and retina.