To be the representative fruition resulted from the rapid development in micro-nano theory and technology, atomic force microscopy (AFM) has greatly promoted the expansion of biological research in micro-nano scale, and facilitated the birth and development of micro-nano biology as an important technique in its 25-year evolutional progress. Based on the fundamental principles and detection modes of AFM, as well as the author’s research findings and work experience in this field, the paper reviews the application of AFM in the study on ultrastructure and biomechanical properties of cells and biomacromolecules in the aspects of biological structure and morphology, surface physicochemical characterization and mechanical manipulation of biological macromolecules, and focus on some important scientific and technical problems on AFM in micro-nano biomedical research needed to be improved and solved urgently, with exploratory insights and recommendations for potential users in ultrastructure and biomechanics of cells and biomacromolecules.