The present disclosure relates to a subfractionation culturing method of a stem cell and proliferation method of a monoclonal stem cell obtained using the same. According to the subfractionation culturing method of stem cells and the proliferation thereof of the exemplary embodiments of the present disclosure, it is advantage that monoclonal stem cells may be quickly obtained without contamination, and desired monoclonal stem cells may be largely obtained in a short time through the rapid proliferation, thereby being used for the preparation of stem cell-therapeutic agents.