Objective To develop a simple and efficient CRISPR/Cas9-based gene editing strategy to broaden the application of human pluripotent stem cell in basic research and regenerative medicine. Methods A CRISPR/Cas9-mediated gene editing approach was established using the lipofection reagent Lipofectamine^TM 3000. With human embryonic stem cell (hESC) as a model, an emerald green fluorescent protein (EmGFP)-P2A-blasticidin reporter module was precisely knocked into hepatocyte nuclear factor 4 alpha (HNF4A) gene. Transfection conditions were systematically optimized, and a dual antibiotic selection strategy was employed to obtained homozygous edited clones, which were validated by Sanger sequencing. The reporter cell line was then subjected to chemical induction toward hepatic differentiation, during which EmGFP expression was detected. Results The method achieved efficient gene knock-in in hESC and yielded homozygous edited clones. During hepatic differentiation, the reporter cell line specifically activated EmGFP fluorescence signals, and its expression was highly consistent with hepatocyte markers albumin and alpha-fetoprotein. Conclusion This study has established a convenient strategy using liposome-mediated transient transfection of CRISPR/Cas9 and donor plasmid vectors, and has achieved a HNF4A-EmGFP-P2A-blasticidin reporter hESC cell line, which can be used to trace hepatic differentiation of hESC.