The invention discloses a method for efficiently separating and purifying mammary epithelial cells. The method is suitable for a plurality of mammals. The mammary tissues of adult mammals, which are in the middle and later periods of gestation or in the lactation period, are collected through aseptic operation. According to the provided method, the separation and purification can be achieved in one step, the whole experiment is simple and efficient; the purification, which comprises repeated differential digestion and differential attachment, in the conventional method is eliminated, the purity of mammary epithelial cells obtained by the provided method can reach 98% or more, the obtained mammary epithelial cells can be continuously cultured in-vitro and passed down for 40 generations, and the lactating function of mammary epithelial cells is not affected. The obtained mammary epithelial cells can be applied to the researches on growth of mammary gland, lactating mechanism, mammary bioreactor, transgenic animals, mammary diseases, and the like.