Two-dimensional (2D) materials are potential candidates for electronic devices due to their unique structures and exceptional physical properties, making them a focal point in nanotechnology research. Accurate assessment of the mechanical and tribological properties of 2D materials is imperative to fully exploit their potential across diverse applications. However, their nanoscale thickness and planar nature pose significant challenges in testing and characterizing their mechanical properties. Among the in situ characterization techniques, atomic force microscopy (AFM) has gained widespread applications in exploring the mechanical behaviour of nanomaterials, because of the easy measurement capability of nano force and displacement from the AFM tips. Specifically, AFM-based force spectroscopy is a common approach for studying the mechanical and tribological properties of 2D materials. This review comprehensively details the methods based on normal force spectroscopy, which are utilized to test and characterize the elastic and fracture properties, adhesion, and fatigue of 2D materials. Additionally, the methods using lateral force spectroscopy can characterize the interfacial properties of 2D materials, including surface friction of 2D materials, shear behaviour of interlayers as well as nanoflake-substrate interfaces. The influence of various factors, such as testing methods, external environments, and the properties of test samples, on the measured mechanical properties is also addressed. In the end, the current challenges and issues in AFM-based measurements of mechanical and tribological properties of 2D materials are discussed, which identifies the trend in the combination of multiple methods concerning the future development of the in situ testing techniques.