【Abstract】 Objective  To establish Doxmycin (Dox) inducible Cas9-genetic editing mouse embryonic stem cell (mESC) line to meet various research requirements in gene editing under different conditions.  Methods According to the principle of the tetracycline (Tet)-on inducible expression regulatory system, the expression plasmid (pCDH-CAG-rtTA-IRES-mCherry) of containing regulatory element reverse tetracy-cline transcriptional activator (rtTA), and the expression plasmid (pTight-Cas9-IRES-GFP-Tight-Puro) of containing Tet-responsive element (TRE) and Cas9 coding sequence, were constructed respectively. The red ?uorescent protein mCherry was ligated with internal ribosome entry site IRES in the rtTA, andgreen ?uorescent protein (GFP) was ligated with IRES in the Cas9, indicating the expression of rtTA and Cas9. Puro resistant gene was constructed for drug selection. The above plasmids were packaged into lentivirus and transfected into mESC in stages, then constructed Dox inducible Cas9-genetic editing mESC Line. Results　Firstly, the inducibility of this system was veri?ed by 293T transient transfection assay. Then, the mESC line with both rtTA and TRE-Cas9 integration were established by combination with report fluorescence and drug selection which appeared the expressed of GFP report after Dox addition. Quantitative polymerase chain reaction assay also showed that Cas9 expression could be successfully induced by Dox.  Conclusion  In this study, a Dox inducible Cas9-genetic editing mESC Line has been successfully constructed, which is more flexibility and controllability in gene editing, and holds high potential application in the future.