The invention relates to a method for culturing mesenchymal stem cells through manual simulation of the bone marrow microenvironment. The method comprises the following steps: acquiring the mesenchymal stem cells, and carrying out culture in vitro in a culture medium by adopting a three-dimensional cell culture scaffold, wherein the three-dimensional cell culture scaffold is made of collagen and chitosan or lysine, or the three-dimensional cell culture scaffold is made of collagen, chitosan and glutaraldehyde. According to the method, by adopting chitosan or lysine, or adopting chitosan and glutaraldehyde to modify the collagen scaffold, the mechanical property of the collagen scaffold can be improved; a three-dimensional porous structure of the three-dimensional cell culture scaffold is adopted for maintaining the three-dimensional growing environment in the in-vitro culturing process of the mesenchymal stem cells, so that the bone marrow microenvironment suitable for the growth of the mesenchymal stem cells can be well simulated, and the problem that in the prior art, the single three-dimensional cell culture scaffold is adopted for culturing the mesenchymal stem cells, the mechanical property of the scaffold is poor, so that the cell proliferation rate is low, and the cell death rate is high, is solved.