Abstract: Objective：To establish a three-dimensional co-culture model of oral cancer cells/fibroblasts and provide a new research method for the follow-up study on the invasion and metastasis of oral cancer. Methods：The method of tissue block was used to isolate and cultivate primary oral mucosa of normal fibroblasts (NFs). NFs and oral cancer cells (Cal27) were used to construct the three-dimensional co-culture model. HE and immunofluorescence staining were used to observe the growth and relationship between Cal27 and NFs of the co-culture model. Results：NFs were successfully harvested by primary culture. Using a floating culture for 4 days, the collagen gel contracted and Cal27 cells showed monolayer growth. When the collagen gel was transferred to the air-liquid interface culture conditions, Cal27 cells showed multilayer growth after 3 days, and the tumor cells exhibited a basement membrane like structure. After 9 days, the basement membrane like structure grew integrated. Conclusions：The three-dimensional air-liquid interface culture model is different from two-dimensional cell culture, which is similar to human tissue structure to form the oral epithelium and the underlying stromal cell layer, so that the oral cancer cells more resemble the human tissue in the growth pattern. Thus it provides a new method and approach for the study of oral cancer.