Abstract: In order to use the Liposome-mediated method to obtain high-level expression of TLR4 gene sheep fetal fibroblast cells as competent donor cells, which was flexible for the in-vitro screen progress to make sure the effective donor cells before embryo transplantation. The preparation of competent donor cell was critical for the production efficiency of transgenic sheep by somatic cell nuclear transfer (SCNT). In this experiment, the ovine fetal fibroblast was transfected by lipofection with the reconstructed vector containing the target TLR4, neo and enhanced green fluorescent protein (EGFP, report gene) genes. Then, they were selected by G418 and detected by EGFP positively. Finally, the stable transgenic ovine fetal fibroblast cell line being over expressed TLR4, was established by identification from both morphological and molecular levels. The results showed that using the optimizing ratio of liposome and the TLR4 vector to go to transfect the fetal sheep fibroblast, then selected and purified, we got 3 different positive clones. Compared the non-transfected fetal sheep cells, the clone of highest expression of TLR4 was transfected in the passage 2, and analyzed by reverse transcription PCR and Real-time fluorescent quantitative PCR, its TLR4 level increased 9.65 times (P<0.01). Collectively, these stable express high level TLR4 donor cells would contributed for breeding new transgenic sheep breed with somewhat anti-disease characteristic, and would be beneficial to provide competent transgenic donor cells for the production of transgenic animal.