In live organisms, cells are embedded in tissue-specific extracellular matrix (ECM), which provides chemical and mechanical signals important for cell differentiation, migration, and overall functionality. Careful reproduction of ECM properties in artificial cell scaffolds is necessary to get physiologically relevant results of in vitro studies and produce robust materials for cell and tissue engineering. Nanoarchitectonics is a contemporary way to building complex materials from nano-scale objects of artificial and biological origin. Decellularized ECM (dECM), remaining after cell elimination from organs, tissues and cell cultures is arguably the closest equivalent of native ECM achievable today. dECM-based materials can be used as templates or components for producing cell scaffolds using nanoarchitectonic approach. Irrespective of the form, in which dECM is used (whole acellular organ/tissue, bioink or hydrogel), the local stiffness of the dECM scaffold must be evaluated, since the fate of seeded cells depends on the mechanical properties of their environment. Careful dECM characterization is also necessary to reproduce essential ECM traits in artificial cell scaffolds by nanoparticle assembly. Atomic force microscopy (AFM) is a valuable characterization tool, as it allows simultaneous assessment of mechanical and topographic features of the scaffold, and additionally evaluate the efficiency of decellularization process and preservation of the extracellular matrix. This review depicts the current application of AFM in the field of dECM-based materials, including the basics of AFM technique and the use of flicker-noise spectroscopy (FNS) method for the quantification of the dECM micro- and nanostructure.