High quality films of polythiophene with different alkyl side chains were successfully synthesized by a novel method in the presence of sodium dodecylbenzenesulonate (SDBS) under N2 atmosphere on the PTFE (polytetrafluorethylene) substrate. The as-prepared films were characterized by scanning electron microscopy (SEM), conductivity measurement, and water contact angle measurement. The morphologies of the films were homogeneous with micro-/nanostructures, and their conductivities were high enough for biomedical applications. Hydrophobicity of the films could be adjusted easily by inducing alkyl side chains with different length, which could control protein adsorption in succession. Hydrophobic polythiophene film with a long alkyl side chain had a higher ability of protein adsorption and PC 12 cell proliferation. The biocompatibility study of the synthesized films in vitro proved that the synthesized films were not cytotoxic to two cell lines used and could support cell attachment and proliferation well. Polythiophenes films prepared by in-situ deposition will be good candidates for biomedical applications.