Objective To observe the three-dimensional morphological characteristics and spatial relationship between the components of rat small intestine based on three different observations: intestinal wall，microvillus and enteraden. Methods A normal SD rat, take the small intestine to produce paraffin specimens. After the processes of serial section and HE staining, digitalized images were carried out in different objective rates. After the processes of assembling,preprocessing,alignment,partition and three-dimensional reconstruction, the spatial relationship between each structure was observed and morphological indexes were measured. Results The three-dimensional configuration and the spatial position of intestinal wall and its internal structures (arteriovenous, lymphatic, microvillus, enteraden, etc) could be displaied intuitirely. Based on the reconstructed image results, some morphological features were measured, which were microvillus density and average height, intestinal density and volume. Conclusion Small intestine submucosa and internal microvillus have abundant vascular nets and lymphatic pipelines, their arrangements are closely related to absorb and immune function of small intestine.