The invention discloses a method for separating sub-totipotent stem cells from human placenta decidua. The invention particularly relates to the technical field of biomedicine. The method comprises the following steps: tissue extraction, pre-digestion treatment, decidua digestion, centrifugal purification, dilution and filtration, sub-totipotent stem cell separation liquid preparation and sub-totipotent stem cell culture preparation. The method utilizes two enzymolysis processes. Enzymolysis is carried out for the first time to remove miscellaneous cells on the amnion, enzymolysis is carried out on the placenta cells for the second time, a disinfecting and washing procedure is carried out once before each enzymolysis operation so that infectious microbe infections are avoided, human sub-totipotent stem cells with high purity are obtained, when the cell suspension is prepared, one-time centrifugal operation is firstly carried out in the last-time enzymolysis operation, and compared withthe traditional two-step centrifugal operation, the third operation is used, so that the sub-totipotent stem cell separation quality is improved, and the preparation efficiency of the human placentasub-totipotent stem cell is ensured.