The mechanical properties of biomaterials play an important role in regulating life processes, and thus accurately delineating the mechanical properties of biomaterials is critical to understand their functionality. Particularly, atomic force microscopy (AFM) has become a powerful and standard tool for characterizing and analyzing the nanomechanical properties of biomaterials, and providing a capability to visualize the thickness of the specimen during AFM-based force spectroscopy experiments benefits the biomedical applications of AFM. Here, we present a study of side-view optical microscopy-assisted AFM based on the integration of AFM and a detachable side-view optical microscopy module, which is able to image in real time the AFM indentation process from the side-view perspective and consequently facilitates the utilization of AFM-based indentation assay to precisely detect the mechanical properties of a specimen by taking its thickness into account. The effectiveness of side-view optical microscopy-assisted AFM was confirmed on four different types of biomaterial systems, including microfabricated structures, hydrogels, living cells, and cell spheroids, and the experimental results significantly show that the mechanical properties of samples at the micro/nanoscale are closely related to their thickness, vividly illustrating side-view optical microscopy-assisted AFM as a promising approach for accurate nanomechanics of biomaterial systems. The study provides additional possibilities for measuring the thickness-dependent nanomechanical properties of biomaterials by AFM, which will enable AFM-based force spectroscopy technology to address more biological issues with enhanced precision and will benefit the field of mechanobiology.