A method for non-enzymatic 3D culture and amplification of mesenchymal stem cells (MSCs) includes the followings steps: preparing PLGA porous microspheres; preparing a PLGA-PEG-PLGA thermosensitive coating microcarrier; culturing and amplifying MSCs; and performing non-enzymatic separation of MSCs, including reducing a culture temperature to below a critical phase transition temperature, and centrifuging a culture medium to collect stem cells. The present invention adopts the method for non-enzymatic 3D culture and amplification of MSCs, wherein the PLGA porous microspheres are used as a cell culture microcarrier scaffold and the thermosensitive hydrogel PLGA-PEG-PLGA is coated on surfaces of such microspheres, without needing additional enzymolysis process, thus efficiently amplifying the stem cells.