Utilizing the zwitterionic alpha-helix peptide bearing a cationic and anionic group at the N- and C-terminus, respectively, we first demonstrated that the vertically aligned multilayer film can be prepared by a simple cast and slow evaporation. The tilt angle of the peptide remained unchanged with ca. 30 degrees in the range between submicrometer and several micrometers in thickness. The key designs allowing simple vertical alignment of the helical peptide multilayer films were (i) monodispersity of the peptide, (ii) electrostatic interaction between anionic substrate and the cationic group hearing at the N-terminus of the peptide, and (iii) interlayer electrostatic interaction among terminal groups of the peptide.