The invention provides a culture medium capable of separating two subgroups of urogenous stem cells. The culture medium is formed by mixing a K-SFM culture medium and a progenitor cell culture medium in an isovolumetric way. The invention also provides application of the culture medium and a separate culture method for human urogenous stem cells. The culture medium and the separate culture method can be used for effectively separating and culturing different subgroups of the human urogenous stem cells and have a good application prospect.