Proteins play a crucial role in regulating life activities and therefore investigating the behaviors of proteins is of critical significance for understanding the underlying mechanisms guiding the physiological and pathological processes of living organisms. Traditional molecular biochemical methods for protein assays are based on ensemble measurements which reflect the averaged behaviors of the whole molecular populations, veiling the rare activities of small molecular subpopulations. Achievements obtained in the past decades have proved that atomic force microscopy (AFM) is a powerful multifunctional tool for characterizing the behaviors of single proteins at work in aqueous conditions with unprecedented spatiotemporal resolution, which provides novel insights into nanoscopic molecular mechanisms and contributes much to the communities of protein biology. In this article, we review the recent advances in AFM-based single-protein analysis from several facets (including morphological imaging, single-molecule force recognition, mechanical unfolding pathways, and high-speed AFM molecular detection), and provide perspectives for future progression.