Here we describe the detailed characterization of gold nanoparticle (Au Np) functionalized surfaces as a biosensing platform by studying a model streptavidin (SA) biotin interaction. Conjugation of SA on Au Np immobilized on silicon (Si) and quartz surfaces and its interaction with biotin were characterized by X-ray photoelectron spectroscopy (XPS), UV-Vis spectroscopy, circular dichromism (CD) spectroscopy, and contact angle measurements. The immobilization method and atomic concentrations of Si 2p, Au 4f, S 2p, C 1s, N 1s, and O is of the resulting SA-biotin modified Si surface were determined by XPS. The CD spectrum and confocal microscopy imaging confirmed that step-by-step modification and bioconjugation can be monitored successfully. Such detailed and well-defined step-by-step characterization provides good information about the surface properties of biosensor platforms. In addition, the LSPR sensing ability of the Au Np based platforms was studied by using a model SA biotin system. A 20 nm spectral red shift was detected when 150 nM SA was immobilized on to the Au Nps surface using the direct incubation/binding method on to the dry surface instead of the flow-injection method. The same platforms were also used to detect the CA 125 antibody antigen system. Large spectral red shifts are very promising in terms of using these surfaces as LSPR biosensors.