Micro-CT, as a non-invasive technology, is widely used in animal experiments to assist in the detection of bone diseases, lung diseases, oral diseases, metabolic diseases, middle and inner ear diseases, tumors and other animal disease models, providing diverse, scientific and reliable image data for animal experiments. It has become one of the indispensable experimental methods in animal experiments. This review will introduce the imaging principles of Micro-CT, review its application in the study of animal disease models, summarize the limitations of Micro-CT technology, and look forward to the future prospects.