Scanning probe microscopy has emerged as a powerful technique for mapping the surface morphology of biological specimens, including proteins and cells. In addition to providing measurements of topographic images, it enables the fabrication of micro-/nanostructures with a high spatial resolution. Herein, we demonstrate a simple and reliable method for the preparation of single Escherichia coli bacterial cell arrays using pre-fabricated microwell structures. Using a < 100 >-oriented silicon substrate, microwell arrays with inclined sidewalls were fabricated by scanning probe lithography and sequential chemical wet etching. The trapping efficiency of single cells was optimized by controlling the geometries of the microwells. These data suggest that single-cell arrays may be applicable in a variety of areas, including drug testing and toxicology, as well as basic cell biology.