To develop a culture substrate that allows efficient expansion of neural stem cells (NSCs), epidermal growth factor (EGF) was immobilized onto the Ni(II)-chelated surface of a glass-based substrate through coordination of Ni(II) to the histidine tag that was fused to the C-terminal of EGF using recombinant technology. For the preparation of the nickel-chelated surface, a thin gold layer was deposited to the glass surface, and then the self-assembled monolayer of alkanethiol terminated with trivalent carboxylic acids was formed on gold and chelated with Ni(II) ions. In the preparation of a monolayer, triethylene glycol-terminated alkanethiol was mixed with carboxylic acid-terminated alkanethiol at various compositions in order to reduce the non-specific adsorption of EGF. The surface analysis of the monolayers was performed by X-ray photoelectron spectroscopy, infrared reflection-absorption spectroscopy, and contact angle measurements. Surface plasmon resonance analyses and protein assays were performed for characterizing EGF-immobilized surfaces. The proliferation and differentiation of rat fetal NSCs were examined on the EGF-chelated substrates to assess quantitatively the effects of alkanethiol composition on the efficiency of stem cell amplification. It was shown that the amplification efficiency was dependent on the alkanethiol composition. This result could be attributed to the difference in the surface density of chelated EGF. Under the optimal condition, 98% of proliferated cells expressed NSC marker. In addition, these cells could be subcultured for further expansion, while retained their multipotency. We concluded that the substrate developed here provides the efficient method for the highly selective expansion of NSCs.