Mechanical forces are always involved in a biological process in which intra- and/or inter-biomolecular interactions are essential. Atomic force microscopy (AFM) is an ideal technique that can be used to investigate the mechanical interactions occurred in biological system, due to its high resolution in force measurement and its capability of working in near-physiological conditions. Single-molecule force spectroscopy based on AFM (AFM-SMFS) has an extraordinary ability to interrogate the intra- and/or inter- biomolecular interactions at the single-molecule and/or single-cell level. In this review, the basic principle of AFM-SMFS, and the techniques including AFM tip modifications (silicon/nitride silicon and gold-coated silicon tips), force spectroscopy measurement and data analysis (the worm-like chain model, the freely jointed chain model and the freely rotating chain model) required in AFM-SMFS, are briefly introduced. The emphasis is given on the recent progress made in investigating biomolecules, including proteins (transforming growth factor beta 1, CD20, Heat Shock Proteins, PTK7, heparin-binding haemagglutinin adhesion and Als5p adhesion proteins) and carbohydrates (glucose, mannose, galactose, group B carbohydrate, capsular polysaccharide, alpha-mannans, beta-mannans, beta-glucans and chitin) existing on mammalian, bacterial and fungal cell surfaces. Finally, the limitations of AFM-SMFS and its future applications have been summarized.