We demonstrate that an Atomic Force Microscope (AFM) can be used to immobilize a di-cysteine-terminated protein (Maltose Binding Protein, MBP-cys-cys for short) at well-defined locations directly on gold substrates via nanografting, and characterize the in situ bioactivity of these proteins within the fabricated nanopatterns. This method exploits the high spatial and orientational control of protein monolayer assembly allowed by nanografting, combined with the high sensitivity of the AFM for detecting ligand-binding events. The maltose-mediated conformational changes within the MBP have been found to change the AFM-tip-protein interaction, therefore causing the frictional signal to change. Our data show that the protein ligand-binding function is maintained upon the immobilization.