The ability to arrange distinct cells in specific, predefined patterns at single-cell resolution can have broad applications in cell-based assays and play an important role in facilitating interdisciplinary research for researchers in various fields. However, most existing methods for single-cell patterning are based on the complicated lithography-based microfabrication process, and require professional skills. Thus, exploiting convenient and universal strategies of single-cell preparation while maintaining high-throughput single-cell patterning remains a challenge. Here, we describe a simple approach for rapid and high-efficiency single-cell patterning using an ultrathin metal microstencil (UTmS) and common tools available in any laboratory. In this work, ultrathin steel microstencil plates with only 5 μm thickness could be fabricated with laser drilling and achieve single-cell prototyping on an arbitrary planar substrate under gravity-induced natural sedimentation without requiring additional fixation, reaction pools, and centrifugation procedures. In this method, the UTmS is reusable and single-cell occupancy could easily reach approximately 88% within 30 min on fibronectin-modified substrates under gravity-induced natural sedimentation, and no significant effect on cell viability was observed. To verify this method, the real-time and heterogeneous study of calcium release and apoptosis behaviors of single cells was carried out based on this new strategy. To our knowledge, it is the first time that a UTmS with 5 μm thickness is directly applied to facilitate the micropatterning of high-resolution single cells, which is valuable for researchers in different fields owing to its user-friendly operation.