One-dimensional nanostructures are ideal building blocks for functional nanoscale assembly. Peptide-based nanofibers have great potential for building smart hierarchical structures due to their tunable structures at a single residue level and their ability to reconfigure themselves in response to environmental stimuli. In this study, it was observed that a pre-adsorbed silk-elastin-based protein polymer self-assembled into nanofibers through a conformational change on the mica substrate. Furthermore, using atomic force microscopy, it was shown that the rate of the self-assembling process was significantly enhanced by applying a nanomechanical stimulus. The orientation of the newly grown nanofiber was mostly perpendicular to the scanning direction, implying that the new nanofiber assembly was locally activated with a directional control. The method developed as a part of this study provides a novel way to prepare a nanofiber patterned substrate using a bottom-up approach.