The present invention relates to a method for isolating and culturing adult stem cells derived from human amniotic membrane in high yield, and more particularly to a method for obtaining a large amount of adult stem cells, the method comprising obtaining amniotic epithelial cells from human amniotic tissue in high yield by treatment with dithiothreitol (DTT) and a low concentration of trypsin and culturing the amniotic epithelial cells in a medium containing a Rho-associated kinase inhibitor. The human amniotic epithelial cell-derived stem cells are easily extracted compared to existing therapeutic stem cells such as umbilical cord blood stem cells and bone marrow stem cells, the yield and proliferation thereof are significantly increased by DTT treatment, the addition of the ROCK inhibitor or the replacement of medium. Thus, the method can be used to efficiently prepare adult stem cells.