The invention discloses a large-scale preparation method of human umbilical mesenchymal stem cell (hUC-MSC). The method comprises the following steps: collecting an umbilical cord and a placenta; separating and extracting the hUC-MSC from the umbilical cord, and carrying out amplification culture by using a traditional DMEM (dulbecco's modified eagle medium)/F12 culture solution containing fetal calf serum (FBS) in the early period; and carrying out hUC-MSC reculture by using the placental homogenate extraction (PHE) with the same source as the umbilical cord instead of the FBS in the later period, thereby obtaining the hUC-MSC raw material cells. On the premise of not using any commercial serum-free culture medium, the method can implement in-vitro large-scale preparation of the hUC-MSC, solves the problem of residues of bovine serum, and saves abundant cost.