Objective To establish the eukaryotic expression vector of Y-box-binding protein 1 (YB-1) with FLAG-tagged and transfect it into hepatocellular carcinoma HepG2 cells to identify the effects of YB-1 on the proliferation and migration. Methods Human YB-1 gene was amplified from the human ovary library by PCR. YB-1 fraction was double enzyme digested and connected with pcDNA3.0-FLAG vector to construct eukaryotic expression vector pcDNA3.0-FlAG-YB-1, which was transfected into HepG2 cells. The expression of YB-1 was detected by Western blotting, and the effect of YB-1 on the proliferation of HepG2 cells was determined by CCK-8 assay and clone formation. The effect of YB-1 on the migration of HepG2 cells was analyzed by wound healing assays. Results The eukaryotic expression vector pcDNA3.0-FLAG-YB-1 was successfully established. YB-1 protein can be expressed in HepG2 cells, and YB-1 promoted the proliferation and migration of HepG2 cells. Conclusion YB-1 promotes the proliferation and migration of HepG2 cells.