Objective: To construction of UPS-E3 ubiquitin ligase ITCH gene Knockout Cells mediated by CRISPR/Cas9. Methods: Three sgRNAs in the PAS domain of the human ITCH gene were designed, constructed of 3 three-in-one plasmids that simultaneously expresses ITCH-sgRNA, Cas9 and eGFP. Recombinant plasmids with correct sequencing sequence were transfected into HeLa cells using Lipofectamine 3000, after 24 hours, inspected the transfection status under a fluorescence inverted microscope. Results: Three ITCH-sgRNAs oligonucleotide single strands were successfully annealed into double strands, after sequencing, it was found that three ITCH-sgRNAs were recombined onto the CRISPR/Cas9 vector. Fluorometric assay showed the green fluorescence was observed in HeLa cells transfected with recombinant plasmids. Conclusions: Successfully constructed three-in-one plasmids that simultaneously expresses ITCH-sgRNA, Cas9 and eGFP, the recombinant plasmids are successfully transfected into HeLa cells, to achieve complete knockout of the ITCH gene in HeLa cells, thus laying the foundation for constructing stable ITCH gene knockout strains.