Self-assembly of amyloid nanofiber is associated with functional and pathological processes such as in neurodegenerative diseases. Despite intensive studies, stochastic nature of the process has made it difficult to elucidate molecular mechanisms for the key amyloid nucleation. Here, we investigated the amyloid nucleation of silk-elastin-like peptide (SELP) using time-lapse lateral force microscopy (LFM). By repeated scanning a single line on a SELP-coated mica surface, we observed sudden stepwise height increases, corresponds to nucleation of an amyloid fiber. The lateral force profiles followed either a worm-like chain model or an exponential function, suggesting that the atomic force microscopy (AFM) tip stretches a single or multiple SELP molecules along the scanning direction, serves as the template for further self-assembly perpendicular to the scan direction. Such mechanically induced nucleation of amyloid fibrils allows positional and directional control of amyloid assembly in vitro, which we demonstrate by generating single nanofibers at predetermined nucleation sites.