This paper describes an atomic force microscope (AFM) based voltage-assisted electrospinning technique. Single nanofibers on substrates are prepared via simultaneous preparation and deposition. In this work, an AFM-based electrospinning process is developed to generate polyethylene oxide (PEO) polymeric single fibers with nanometer scale diameters. The results demonstrate the feasibility of this developed approach for assembling nanofibers at predetermined positions. This work represents a promising advancement in nanomanufacturing of one-dimensional nanostructured materials for micro- and nanoscale devices.