Abstract: Objective The study was to construct and identify the eukaryotic expression plasmid pEGFP-C3/Smad3, and then transfect it into MCF-7 breast cancer cells.To lay a foundation for further research on the biological function of Smad3 in the inhibition of breast cancer growth mechanism.Methods The cDNA was extracted from MCF-7 breast cancer cells by RT-PCR, the Smad3 gene fragment was amplified and extracted by PCR, and the amplified products were inserted into pEGFP-C3 eukaryotic expression vector then sequenced and identified by restrictive endonuclease digestion.The constructed recombinant plasmid was transitorily transfected into the MCF-7 breast cancer cells and the expression of Smad3 was observed by inverted fluorescence microscope, RT-PCR technology and westen blot.Results The experiment was successfully constructed the eukaryotic expression plasmid pEGFP-C3/Smad3 and transiently transfected into MCF-7 cells.Conclusion The eukaryotic expression plasmid pEGFP-C3/Smad3 was transiently transfected into MCF-7 cells.Smad3 gene and protein levels can be significantly raised.