Immobilization of proteins in a functionally active form and proper orientation is fundamental for effective surface-based protein analysis. A new method is presented for the controlled and oriented immobilization of ordered monolayers of enzymes whose interaction site had been protected using the protein ligand. The utility of this method was demonstrated by analyzing the interactions between the enzyme ferredoxin-NADP+ reductase (FNR) and its redox partner ferredoxin (Fd). The quality of the procedure was deeply evaluated through enzymatic assays and atomic force microscopy. Single-molecule force spectroscopy revealed that site-specifically targeted FNR samples increased the ratio of recognition events 4-fold with regard to the standard randomly modified FNR samples. The results were corroborated using the cytochrome c reductase activity that gave an increase on surface between 6 and 12 times for the site-specifically targeted FNR samples. The activity in solution for the enzyme labeled from the complex was similar to that exhibited by wild-type FNR while FNR randomly tagged showed a 3-fold decrease. This indicates that random targeting protocols affect not only the efficiency of immobilized proteins to recognize their ligands but also their general functionality. The present methodology is expected to find wide applications in surface-based protein-protein interactions biosensors, single-molecule analysis, bioelectronics or drug screening.