The design and initial characterization of the self-assembled gold colloid monolayer by a sandwich structure via the immunological identification are reported. The 13 nm gold colloid nanoparticles and the silicon or quartz substrates have been modified with the mouse polyclonal antibody against hepatitis B virus surface antigen (PAb) and the mouse monoclonal antibody against hepatitis B virus surface antigen (MAb), respectively. They can be linked by a special reaction with their corresponding hepatitis B virus surface antigen (Antigen) as a sandwich structure. Thus, the density of gold nanoparticles self-assembled on the substrate can be readily controlled by the amount of the antigen added. The resulting substrates have been characterized by atomic force microscopy (AFM) and surface-enhanced Raman scattering (SERS) spectroscopy when the gold nanoparticles were modified with SERS-active probe molecules of 4-mercaptobenzoic acid (MBA) after silver enhancement. These data show that the gold nanoparticles are separately fixed onto the substrate and form a uniform monolayer, which possess a set of features that make them very attractive for both basic and applied uses, including roughness, high stability, and biocompatibility.