The whole-genome sequence of the olive flounder (Paralichthys olivaceus) provides a basis for gene functional analyses,which is important for the aquaculture industry.Understanding gene function will help us to select better economic traits such as fast growth and better culture conditions,which further will increase the aquaculture output.Gene knockout is an important reverse ge-netics approach for in vivo studies of gene function.In this study,the CRISPR/Cas9 genome editing method with a microinjection sys-tem using a simple braked needle was employed in olive flounder.After injection in embryos,green fluorescent protein expression was detected in 40％ of larvae.The proportion of normal-hatched larvae was approximately 50％.Different mutations,including short indels and fragment deletions,were found in our test genes gsdfand myomaker.Additionally,we detected more than one mutation in a single larva.In summary,our microinjection technique and CRISPR/Cas9 can be applied to study gene functions in olive flounder.