Rationale: Accurate diagnosis and understanding of gastrointestinal (GI) diseases such as ulcerative colitis and Hirschsprung's disease remain challenging due to the limitations of traditional two-dimensional (2D) histopathology in capturing the intricate three-dimensional (3D) architecture and dynamic microenvironment of GI tissues. This study explores the potential of integrating 3D imaging techniques with artificial intelligence (AI)-based analysis to improve histological evaluation and diagnostic accuracy. Methods: Using advanced imaging and computational tools, we identified critical structural and functional details of the enteric nervous system and associated tissues that are often missed by 2D approaches. Results: The results showed that 3D imaging coupled with AI significantly improves diagnostic accuracy and provides new insights into disease mechanisms, enabling earlier and more precise detection of pathological changes. In addition, this approach enhances our understanding of the pathophysiology of GI diseases, bridging gaps in both clinical and basic research. Conclusions: These findings underscore the transformative potential of 3D imaging and AI to revolutionize diagnostic workflows and advance our knowledge of GI diseases, ultimately contributing to improved patient outcomes and innovative research methodologies.