Convenient preparation of nano/micro scale topography is crucial for the fabrication of low-cost biodevices, which could be useful tools for understanding cell biology mechanisms and for the development of scaffolds for tissue engineering. Such intelligent surfaces have been conventionally fabricated through photolithography, micro-contact printing, and nano/micro imprinting. However, considering the process integration, these approaches are not always adequate in order to produce large dimensional patterns in a convenient and rapid way. In this study, we focused on the convenient fabrication of nano-wrinkles based on the elastic instability between a shape memory polymer sheet and a conductive polymeric film, on which the behavior of murine skeletal muscle cells (C2C12) was evaluated. A tens-of-nm-thick layer of poly(3,4-ethylenedioxythiophene) with poly(styrenesulfonate) (PEDOT:PSS) was spincoated on a thermo-retractable polymer sheet. Then, thermal treatment produced different periodicity of the unidirectional nano-wrinkles on the polymer sheet covered with different thickness of PEDOT:PSS layer. Finally, adhesion and proliferation of C2C12 were evaluated, comparing different samples. The cells preferentially adhered and anisotropically aligned on low and narrow ridges (1.5 μm height) rather than on high and wide ones (2.5 μm height). Furthermore, we observed that these trends were confirmed in the differentiation stage of C2C12 into myotubes. The combination of living cells and tunable nano-wrinkles made of conductive polymeric materials will represent a unique tool for the development of innovative biomedical devices.