The invention relates to a construction method of a human amniotic mesenchymal stem cell bank. The construction method comprises the following steps: taking a human amnion for detection, flushing and washing the human amnion with a phosphate buffered solution, then smashing the human amnion, diluting with the phosphate buffered solution, digesting with trypsin, digesting with collagenase IV and deoxyribonuclease I, and filtering to obtain a single-cell suspension; by adding human serum albumin, transferring, insulin and sodium selenite into a DMEM/F12 basal culture medium with the ratio of VDMEM to VF12 being 1 to 1, placing human amnion mesenchymal stem cells in an incubator under the serum-free condition, and then performing liquid exchange and culture transfer; subjecting the mesenchymal stem cells obtained through in vitro culture and proliferation to liquid nitrogen refrigeration, and preserving the cells according to the gender of newborn infants, the ABO/Rh type and the HLA type; establishing cell information files, so that the human amniotic mesenchymal stem cell bank is constructed. The method has the characteristics of no other animal derivation, wide source range and no ethic limitation. With adoption of the method, the human amniotic mesenchymal stem cells can be provided for cell therapy and other application.