Biotin-capped gold nanoparticles assembled on flat gold with volume fraction f are studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM) in order to estimate the dielectric function of the gold nanoparticles based on the Maxwell−Garnett (MG) theory. The complex dielectric function (ε‘, ε‘‘) of the spherical nanoparticles at three representative wavelengths in the vis−near-IR region, i.e., λ = 543, 632.8, and 1152 nm, is estimated for a surface homogeneously covered with nanoparticles in order to discuss the wavelength dependence of the dielectric function. The SPR response of a surface covered with particles in 2D aggregates is also analyzed. The experimental SPR curve of the particle aggregates deviates from the theoretical predictions, suggesting dipole interactions between particles.