Objective To establish a small intestinal organoid culture system as an in vitro study model of intestinal epithelial cells, and to explore the relevant pathological detection techniques and provide a convenient platform for in vitro study of various intestinal diseases. Methods The mouse intestinal epithelium was isolated and cultured into organoids to simulate the growth and development of intestinal epithelium in vitro. The proliferation and differentiation signals were detected by immunohistochemistry and three-dimensional immunofluorescence technique.Results The culture system of the mouse small intestine epithelium was established. Immunohistochemical staining and three-dimensional immunofluorescence technique were successfully used to detect the growth and development of small intestinal organoids.Conclusions The successfully established mouse small intestinal organoid culture system and application of immunoassay technology will gradually become a most favorable technical means for studies of various intestinal diseases.