[Objectives]  To investigate the ultrastructure of the midgut of Drosophila melanogaster using scanning electron microscopy and three-dimensional reconstruction, thereby improving understanding of its function. [Methods]  Midguts were removed from wild-type D. melanogaster, continuously sectioned and prepared for scanning electron microscopy using the osmium tetroxide-thiocarbohydrazide-osmium tetroxide (OTO) method. The ultrastructure of midgut tissue was then observed under a scanning electron microscope. Specific structures of the midgut were reconstructed in 3D with Amira software. [Results]  Midgut ultrastructure consisted of a serosal barrier, muscle layer, perinatal membrane, mucous layer, intestinal microorganisms, tracheole, intestinal epithelial cells, intestinal stem cells and enteroendocrine cells. Intestinal epithelial cells contain mitochondria, microvilli and basal labyrinths. A three-dimensional reconstruction of the intestinal microvilli, mitochondria, luminal contents, microtrachea and basal labyrinths, was created. [Conclusion]  3D reconstruction was an effective way to accurately and comprehensively visualize the ultrastructure of the D. melanogaster midgut.