The interlayer bonding in two-dimensional (2D) materials is particularly important because it is not only related to their physical and chemical stability but also affects their mechanical, thermal, electronic, optical, and other properties. To address this issue, we report the direct characterization of the interlayer bonding in 2D SnSe using contact-resonance atomic force microscopy (CR-AFM) in this study. Site-specific CR spectroscopy and CR force spectroscopy measurements are performed on both SnSe and its supporting SiO2/Si substrate comparatively. Based on the cantilever and contact mechanic models, the contact stiffness and vertical Young’s modulus are evaluated in comparison with SiO2/Si as a reference material. The interlayer bonding of SnSe is further analyzed in combination with the semi-analytical model and density functional theory calculations. The direct characterization of interlayer interactions using this non-destructive methodology of CR-AFM would facilitate a better understanding of the physical and chemical properties of 2D layered materials, specifically for interlayer intercalation and vertical heterostructures.