In this work, we designed a simple and low cost acid-dissolved/alkali-solidified self-sphering shaping method (AASS) to automatically fabricate three-dimension (3D) stem cell expansion microsphere scaffolds in large scale to satisfy the urgent need for stem cells in tissue engineering and clinical medicine research. We chose chitosan as the main part of microsphere scaffold, graphene oxide of 3 wt% as the strength agent, and genipin as the fluorescence generator to fabricate fluorescent chitosan (CS)/graphene oxide (GO) hybrid microspheres. The diameters of the hybrid microspheres are about 400 mu m with the diameter error less than 10%, which is suitable for stem cells spreading. These hybrid microspheres with good biocompatibility can support the cells' spread, growth and proliferation. After cultured for 5 days, the total number of cells on microspheres has almost increased fourfold. Most importantly, the microspheres can maintain the cell type. After cultured for 7 days, almost all cells still express main markers of human umbilical cord mesenchymal stem cells (HUMSCs). The hybrid microspheres can support long-time stem cell expansion because of their good mechanical strength, controllable degradability, and low expansion rate when soaked in media. Furthermore, their autofluorescence also makes observing and tracking the stem cells behavior on surface of microsphere scaffolds more convenient. This research provides a powerful method for mass-producing chitosan/graphene oxide hybrid microspheres for 3D stem cells expansion. This method is easy to be put into industrial production, and may have tremendous value in medicinal and clinical applications.