Intestinal tract is the major organ for digestion and absorption of food nutrients. The establishment of an effective intestinal ex vivo model is helpful to promote the development of a novel evaluation system for absorption and efficacy of food nutrients and functional active substances. Based on the intestinal crypt containing intestinal stem cells， we created an integrated set of organoid culture of different intestinal segments (including duodenum， jejunum and ileum) of mouse through optimizing the isolation method of intestinal crypt from different intestinal segment and the composition of culture medium. In addition， RT-PCR and immunofluorescence results demonstrated that the organoids derived from different segment recapitulate the structural characteristics and cell composition of corresponding intestinal tissues. These characters had high stability during in vitro repeated passage. In short， the establishment of orgnaoid culture of different intestinal segment may provide a promising ex vivo model for the evaluation of absorption and function of food nutrients in the food research field.