Objective：To establish stable and efficient method for primary culture of human palatine tonsil crypt epithelial cells. Methods：Eighty palatine tonsil tissues were derived from children aged from 3 to 5 undergoing surgical removal of tonsil. Tissue piece method and combined type Ⅱ dispase and 0.25% trypsin-EDTA digestion method were used to separate primary palatine tonsil crypt epithelial cells，and the effectiveness of two isolation methods were compared. Serum-free Keratinocyte medium were applied to purification and primary cultivation. Growth feature and morphology of primary cells were observed under an inverted microscope. Immunofluorescence and immunocytochemistry technique were used to identify the specification of primary cell. Results：The success rate，density and primary cell fusion time of the dispase digestion group were all higher than those of the tissue piece group（P < 0.05）. The primary tonsil crypt epithelial cell began adherent growth after 2 days isolation from the tonsil tissues. The shape of cell was polygon under microscope，and cells connected like islands. Primary cells formed confluent monolayers after 12 days’ cultivation，it seemed like paving stone. Pancytokeratin positive staining was determined by immunocytochemistry. The specific cytokeratin 8/18 staining was positive after immunofluorescence identification. Conclusion：It is an efficient and repeatable method for primary isolation and cultivation of human palatine tonsil crypt epithelial cells by using combined type Ⅱ dispase and 0.25% trypsin-EDTA digestion method with serum-free keratinocyte medium.