Objective:To apply CRISPR/Cas9 technology to construct stable OSBPL2 gene knockout HeLa strain. Method:Three single-guide RNA(sgRNA) targeting respectively to exon 2,3,5 of OSBPL2 gene were designed, then 3 recombinant eukaryotic expressional plasmids by the carrier of PGK1.1 were constructed. After transfection to HeLa cell respectively, puromycin was performed to screen positive cells and then cruiserTM gene knockout detection in combination with the sequencing were used to analyze targeting effect of the three plasmids. HeLa cells with the plasmid of the optimal targeting effect continued to screen monoclonal cell. The knockout effect was measured by Western blot. Results: SgRNAs were correctly inserted into the recombinant plasmids, OSBPL2 protein was undetected in HeLa cell after transfection with plasmid targeting to exon 2 and screening of monoclonal cell. Conclusion:Stable OSBPL2 gene knock-out HeLa strain can be successfully built.