Amyloid fibrillogenesis is a pathogenic protein aggregation process that occurs through a highly ordered process of protein-protein interactions. To better understand the protein-protein interactions involved in amyloid fibril formation, we formed nanogold colloid aggregates by stepwise additions of ∼2 nmol of amyloid β 1-40 peptide (Aβ1-40) at pH ∼3.7 and ∼25 °C. The processes of protein corona formation and building of gold colloid [diameters (d) of 20 and 80 nm] aggregates were confirmed by a red-shift of the surface plasmon resonance (SPR) band, λpeak, as the number of Aβ1-40 peptides [N(Aβ1-40)] increased. The normalized red-shift of λpeak, Δλ, was correlated with the degree of protein aggregation, and this process was approximated as the adsorption isotherm explained by the Langmuir-Freundlich model. As the coverage fraction (θ) was analyzed as a function of ϕ, which is the N(Aβ1-40) per total surface area of nanogold colloids available for adsorption, the parameters for explaining the Langmuir-Freundlich model were in good agreement for both 20 and 80 nm gold, indicating that ϕ could define the stage of the aggregation process. Surface-enhanced Raman scattering (SERS) imaging was conducted at designated values of ϕ and suggested that a protein-gold surface interaction during the initial adsorption stage may be dependent on the nanosize. The 20 nm gold case seems to prefer a relatively smaller contacting section, such as a -C-N or C═C bond, but a plane of the benzene ring may play a significant role for 80 nm gold. Regardless of the size of the particles, the β-sheet and random coil conformations were considered to be used to form gold colloid aggregates. The methodology developed in this study allows for new insights into protein-protein interactions at distinct stages of aggregation.