Objective：To construct over-expressing pCAG-3×flag-PP2A Cα transgenic mice，screen the stable system of high level expression，and therefore to provide a transgenic animal model for the study of the biological function of PP2A Cα. Methods：The pCAG-3×flag-PP2A Cα transgenic vectors were constructed by inserting mice PP2A Cα cDNA and Flag protein sequence tag to the downstream of pCAG promoter. The linear pCAG-3×flag-PP2A Cα plasmid was injected into the zygote nucleus by microinjection technique to get the over-expressing pCAG-3×flag-PP2A Cα mice. The positive over-expression mice were identified by PCR. The positive expression of total protein in mice was extracted and the expression of transgenic mice was analyzed at the protein level. Results：The pCAG-3×flag-PP2A Cα transgenic vector and transgenic mice was successfully constructed by PCR and was verified by plasmid sequence analysis. The system of high level expression transgenic mice was screened by Western blot assay. Conclusion：The pCAG-3×flag-PP2A Cα transgenic mice with stable system of high level expression were screened and obtained.