Milk contains heterogeneous cell populations such as mammary epithelial cells, immune cells, and pluripotent stem cells. These cells not only participate in the synthesis and secretion of nutrients but also play roles in immune defense, tissue repair and development, directly affecting the quality of milk and the health of mothers and infants. The emergence of single-cell RNA sequencing (scRNA-seq) technology enables researchers to map the milk-derived cell atlas at the single-cell level, revealing the lactation regulation network and the mechanism of cell-cell interaction. This technology provides a new tool for optimizing the quality of milk sources and maternal and infant interventions. This review mainly summarizes the major cell types and their functions in human and bovine milk, as well as the current status of the application of scRNA-seq technology in milk-derived cells, providing a reference for further studies on the lactation mechanism of milk-derived cells.