Two-dimensional, crystalline bacterial cell surface layers, termed S-layers, are one of the most commonly observed cell surface structures of prokaryotic organisms. In the present study, genetically modified S-layer protein SbpA of Bacillus sphaericus CCM 2177 carrying the short affinity peptide Strep-tag I or Strep-tag II at the C terminus was used to generate a 2D crystalline monomolecular protein lattice on a silicon surface. Because of the genetic modification, the 2D crystals were addressable via Strep-tag through streptavidin molecules. Atomic force microscopy (AFM) was used to investigate the topography of the single-molecules array and the functionality of the fused Strep-tags. In high-resolution imaging under near-physiological conditions, structural details such as protein alignment and spacing were resolved. By applying molecular recognition force microscopy, the Strep-tag moieties were proven to be fully functional and accessible. For this purpose, streptavidin molecules were tethered to AFM tips via approximately 8-nm-long flexible polyethylene glycol (PEG) linkers. These functionalized tips showed specific interactions with 2D protein crystals containing either the Strep-tag I or Strep-tag II, with similar energetic and kinetic behavior in both cases.