Medaka (Oryzias latipes) is one of the model organisms for studying gene function, organogenesis, and development mechanism. Although editing technology has been widely used in medaka, there was no detailed report on the use of CRISPR/Cas9 to establish O. latipes mutant. To thoroughly describe the establishment process of O. latipes mutant, this study takes the knockout of Olpax6.1 as an example. Firstly, the gRNA target of Olpax 6.1 gene was designed using the ChopChop website; secondly, the template for in vitro transcription of gRNA and Cas9 mRNA were obtained by PCR and plasmid digestion respectively, and gRNA and Cas9 mRNA were synthesized by in vitro transcription; thirdly, the mixture of Cas9 mRNA and gRNA was microinjected into medaka embryos to obtain the mutated F₀; finally, we used PCR, T7 Endonuclease I digestion and Sanger sequencing to screen the offspring of the cross between F₀ and wild type to obtain F₁ with the same mutation type, and then crossed the same mutated F₁ and screened their offspring to obtain Olpax6.1 knockout homozygous mutant, which displays the classic phenotype of ocular developmental malformations. Taken together, this study provides technical guidance for the generation of mutants in O. latipes and other fish.