With the rapid development of polymer materials, the simultaneous acquisition of micro-nano structure and mesoscopic mechanical properties from the material surface with the transverse resolution of nanoscale has become a hot spot in the research of polymer materials. Atomic force microscopy-based highresolution imaging and single-molecule force spectroscopy techniques have been widely used in various related fields. This review introduces the basic design and working principles of atomic force microscopy as well as single-molecule force microscopy and their applications in the characterization of polymer surface morphologies, polymer film phase separation, polymer crystallization behavior, polymer chain stretching, protein folding/unfolding, G quadruplex intermolecular and intramolecular interactions, living cell surface proteins, and sugars. The research status of AFM probe modification strategies such as nanomaterial processing, single molecule manipulation and biomolecule etching is reviewed, finally, the limitations and improvement potential of single molecule force spectroscopy based on atomic force microscopy (AFM- SMFS) are summarized and discussed, and the new research practice in the field of polymer materials is prospected.