Layer-by-layer self-assembly ultrathin films continue to be of significant interest among researchers for its broad application in diverse areas. Based on electrostatic attraction between materials with opposite charges, multilayer films of (PSS/Cyt c) 6, (GNPs/Cyt c) 5 and (PDDA/DNA) 6 had been successfully fabricated on the surface of gold chip, respectively. The adsorption process had been followed by in suit spectral surface plasmon resonance (SPR) technique in real time. The experimental results demonstrate that adsorption of materials onto the gold chip can induce a redshift of resonance wavelength and the amount of adsorption can be determined by the change of resonance wavelength. Kinetics studies suggest that adsorption of cytochrome c (Cyt c) or deoxy ribonucleic acid (DNA) obeys Langmuir-isotherm theory, while adsorption of gold nanoparticles (GNPs) obey diffusion-controlled model. These results also indicate that GNPs need more time to reach adsorption equilibrium than Cyt c and DNA, due to a small value of diffusion coefficient.0 increasing with the layer, however, (GNPs/Cyt c) 5 and (PDDA/DNA) 6 show a nonlinearly increase. Moreover, through controlling the number of assembled layer, the thickness of multilayer films can be precise designed.