The present invention relates to a method for co-culture of stem cells using feeder cells, more particularly to a method for culturing stem cells by using a membrane having a number of pores to separate stem cells and feeder cells. In the present invention, the culture condition of stem cells optimized is provided, in which stem cells and feeder cells are cultivated independently in separate spaces while permeating essential substances selectively. The stem cells prepared in the present invention continue to remain indifferent and be supported by feeder cells until needing being sub-cultured. In addition, the stem cells even for therapeutic use can be obtained without any contaminant since not pretreated with a cytostatic agent such as mitomycin or irradiated. Therefore, the method for co-culturing stem cells by using a membrane of the present invention can be widely used for clinical applications.