This paper reports a new method of producing electrospun zein/polyvinyl alcohol (PVA) mats as three-dimensional (3D) cell culture surface material. The electrospun structure has many advantages and protein-based biomaterials possess unique properties preferred for cell biocompatibility. However, electrospun fiber matrices developed from proteins have poor mechanical properties and morphological stability in the aqueous environments required for cell culture. Efforts have been made to improve the properties of electrospun protein scaffolds, including dry mechanical for handling and surface hydrophilicity, but the current methods have major limitations, such as cytotoxicity and low efficiency. In this research, experimental data showed that the adding different proportion of PVA influenced the properties of the zein mats. Considering the properties of the mats and mouse embryonic stem cells growth behavior on the various electrospun mats, the EM3 (zein/PVA with mass ratios of 3:1) showed better growth status than any other mats. EM3 affixed to cover-glass could be autoclaved for 30 min at 120 degrees C. In addition, embryonic stem cells cultured on chosen electrospun zein/PVA mats in vitro proved that culture products were easily attached onto mat and mat could be used for induction of stem cell differentiation and therefore promising mats for 3D cell culture surface applications.