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Single-cell analysis has been considered as a promising way to uncover the underlying mechanisms guiding the mysteries of life activities, which considerably complements traditional ensemble assays and yields novel insights into cell biology. The advent of atomic force microscopy (AFM) provides a potent tool for investigating the structures and properties of native biological samples at the micro/nanoscale under near-physiological conditions, which promotes the studies of single-cell behaviors. In the past decades, AFM has achieved great success in single-cell observation and manipulation for biomedical applications, demonstrating the excellent capabilities of AFM in addressing biological issues at the single-cell level with unprecedented spatiotemporal resolution. In this article, we review the recent advances in single-cell analysis that has been made with the utilization of AFM, and provide perspectives for future progression.