Dynamic re-organization of individual adhesion nanoclusters in living cells was demonstrated using ligand-patterned surfaces. The method was based on a strategy in which ICAM-1 ligands immobilized in chemically confined patterns were presented to monocytes in a controlled manner to induce a spatio-temporal reorganization of LFA-1 nanocluster on the cell side. Single-molecule detection along with TIRF microscopy was used to visualize the molecular diffusion at the nanometer scale. Immobilized ligands accelerated the direct comparison of their distinct spatial and temporal landscape and diffusive behavior. Microcontact-printing (MCP) technique was employed to fabricate tailored ICAM-1 surfaces. The fabrication process involved transfer of goat-anti-human fragments into the glass substrates using MCP, blocking the unexposed glass surface with bovine serum albumin (BSA), and binding of chimera-Fc-ICAM-1.