The invention discloses a preparation method of a post-resuscitation protective agent for refrigerated umbilical cord blood hematopoietic stem cells, and aims to solve problems that in existing pre-treatment of resuscitating the refrigerated umbilical cord blood hematopoietic stem cells by using normal saline, the cells are easy to be agglomerated, cell debris is increased, a survival rate is reduced and the number of the cells is reduced. The method comprises the following steps: one, preparing buffer solution; two, preparing plasma without fibrin; and three, adding low-molecular-weight heparin sodium, hydroxyethyl starch and polyethylene glycol to the buffer solution; and finally adding the plasma without fibrin, completing. The protective agent is capable of effectively protecting the survival rate of the cells, wherein the cells are not easy to be agglomerated, and cell colony formation is better, effectively protecting activity of the umbilical cord blood hematopoietic stem cellsafter resuscitation, and the number of the cells, wherein the cells are not agglomerated, and guaranteeing a formation force of a colony. The protective agent is applied to the preparation field of the post-resuscitation protective agent for the hematopoietic stem cells.