Three-dimensional (3D) organoids act as model systems because they mimic in vivo tissue morphology. Recent advancements in the field have demonstrated that organoids derived from various organs have assisted in understanding the underlying mechanisms of disease modeling and expanded our knowledge of organ development in vitro. Furthermore, these organoids have become a promising biomaterial in regenerative medicine for therapeutic purposes as well as in nutritional research for feed efficiency measurement in livestock. Intestinal organoids of livestock, including pigs, cattle, chickens, and horses, have been developed. These could be used to examine host-pathogen interactions, such as interaction between enteric viruses and epithelial cells, and are potential alternatives to in vivo systems. However, there are very limited studies regarding species-specific medium to cultivate and establish intestinal organoids of livestock. Species-specific medium is applied differently between species for the cultivation of intestinal organoids, and its modification is important for the maintenance of specific cell types or genes from the cellular diversity of the intestinal epithelium. In this study, we introduce the histological development of a 3D culture system and a species-specific medium for the cultivation of intestinal organoids in livestock. Finally, we discuss the importance and future perspectives of intestinal organoids in the fields of agriculture and biotechnology for various purposes.