Objective: To inspect the feasibility of recombinant stable HEK293 cell lines development for biopharmaceuticals production using CRISPR/Cas9-mediated site-specific integration. Results: Using EGFP as a model protein, we first confirmed that the ‘safe harbor’ AAVS1 locus could be successfully targeted and the exogenous genes could be integrated through homology-directed repair induced by CRISPR/Cas9 technology. Then we constructed a donor plasmid harboring CTLA4Ig gene with an upstream CMV promoter and a downstream puromycin N-acetyltransferase gene to accelerate the efficient integration and selection of CTLA4Ig expression clones. After puromycin enrichment, the transfected pool was diluted for single clone selection, and 12 recombinant clones with CTLA4Ig expression were finally selected with a targeting efficiency of 25.8%. Productivity assay demonstrated that a frequency of 83.3% of selected clone were of consistent productivities, thus illustrating the high efficiency and success rate of this strategy. Conclusions: CRISPR/Cas9 mediated site-specific integration is an efficient and reliable tool to establishment recombinant stable HEK293 cell lines for both academic and industrial applications.