The invention discloses a method for isolated culture of esophagus epithelial stem cells. The method comprises the steps of taking esophagus mucosa tissue and epithelium adhering to the esophagus mucosa tissue, removing muscular tissue, taking a basal layer of epithelium mucosae tissue, shearing the basal layer of epithelium mucosae tissue into bits, conducting digestion centrifugation, and spreading obtained cells in a culture flask, culture plate or culture dish coated with a matrix; then adding a DMEM/Ham's F-12 culture solution containing HEPES, sodium bicarbonate, L-glutamic acid, FBS, EGF, insulin, hydrocortisone, vibrio cholerae toxin, transferring, BPE, quasi-vitamin A and cell signaling pathway inhibitor; finally, placing the culture flask, culture plate or culture dish containing cell suspension in a 37 DEG C incubator for culture, and obtaining the single-layer epithelial stem cells in 10-14 days. According to the method, operation is convenient, safety performance is high, the esophagus epithelial stem cells are obtained through separation, the activity of the stem cells is high, and application prospects in tissue engineering and regenerative medicine are broad.