We report the peptide-directed preparation and X-ray structural study of biofunctionalized Au nanoparticles (NPs) deposited on Ti surfaces. Au NPs were prepared by reduction of Au(3+) compound onto HCl-refreshed Ti in the presence of thiol-functionalized small peptides. A modified extended X-ray absorption fine-structure (EXAFS) technique, equipped with a rotating-stage and glancing-angle setup, was able to more sensitively detect the structure and bonding of Au NPs on Ti with low surface coverage. It was found that the use of the tripeptide glutathione (GSH) results in smaller NP size when compared to N-(2-mercapto-propionyl) glycine (MPG), a pseudodipeptide, over a wide range of Au/peptide molar ratios (20:1, 10:1, 5:1, and 2:1). By varying the ligand concentration, the Au NP structure in both systems can be controlled, generating nanocrystals, nanoclusters, and Au-thiolate polymer, which is unique for substrate-supported NP synthesis. This work presents a facile preparation of Au-peptide nanoparticles on biocompatible surfaces, and illustrates the high sensitivity of this modified EXAFS technique for structural studies of substrate-supported nanoparticles with low coverage.