Abstract: Tissue mass inoculation method was used in this study, and mammary epithelial cells of diary cow were successfully cultivated, mammary gland epithelial cells were identified using cytokeratin 18 immunofluorescence staining. The survival rate of cells inoculation，doubling time of cell population, growth curve，morphological and biological character were detected，and identification and establishment of normal cultured dairy cow mammary epithelial cell lines were set up，cell could be passaged 20 generations with a good behavior of proliferation. Through the cell passage and repeatedly freeze-thaw， dairy cow mammary gland epithelial cell lines were established, and a large number of mammary epithelial cells were acquired.