Objective To reduce the animal component contamination for human embryonic stem cells (hESCs) and to simplify hESCs culture process, we develop a new coating substrate which can support the hESCs growth without differentiation, and is easy to store and use. Methods Mouse embryonic fibroblasts(MEF)were fixed on the surface of plate by methanol. hESCs were cultured on this new substrate and were passaged every 5 to 6 days. After 10 passages, we checked the cell morphology, alkaline phosphatase expression, embryonic specific markers and the differentiation ability in vitro. Results After 10 passages, the hESCs grew well on this new substrate and maintained the typical hESCs morphology. Alkaline phosphatase staining was positive. Immunofluorescence staining showed that the expressions of Oct4, SSEA4, Tra-1-60 were positive. The cells formed embryoid body in vitro. Conclusions This methanol-fixed MEF substrate can support the growth of undifferentiated hESCs. The coating material can be produced in large scale and stored for a long time. It provides a new and relatively easy way to amplify hESCs.