In recent years, with the introduction of the concept of metagenomics, human metagenomics has also become a new approach to reveal the development and health of humankind. As the largest microflora in the human body, the gut microbiome has been found to play an important role in the normal physiological functions of the human body and the occurrence and development of diseases. The variety and quantity of gut microbiome are abundant. In studies of the gut microbiome, the application of germ-free animals makes the experiments more accurate and convenient. It is an ideal basic animal model, which is of great significance to the research of microecology. This review summarizes the current understanding of germ-free animals, the application of germ-free animals in experimental research, and the prospects of germ-free animal models to review the application and development of germ-free animals in research of the gut microbiome.