The lactating mammary glands in ruminants play a pivotal economic role in agriculture and food nutrition. The need for an improved 3D in vitro model to study its lactating biology highlights the importance of ongoing research in this field. Overcoming these challenges represents a crucial area of research, with the development of protocols for livestock lactating organoids being a significant goal for the future. In this study, we utilized virgin dairy goats as a model and successfully established a mammary organoid system. These organoids, cultured within an extracellular matrix (ECM) gel, maintained a bilayer structure that closely resembled the original mammary tissue architecture. Importantly, the expression patterns of genes related to fatty acid synthesis and milk proteins in lactating organoids closely mirrored those observed in lactating mammary tissues. Proteomic analysis further confirmed that major milk proteins were highly produced in the lactating organoids. In summary, this study successfully developed a mammary organoid culture system that holds significant promise as a novel platform for cell-based milk production.