Small-angle X-ray scattering and UV-vis absorption measurements were performed on mixed solutions of gold nanoparticles (AuNPs) and atelocollagen (AC), triple-helical collagen without telopeptide, in acetate buffer at pH 4 under different temperature conditions, i.e., preparation temperature Tprep and measurement temperature Tmeas. Due to the significantly higher electron density of gold than that of AC, the structure factor S(q) of AuNPs is readily estimated from the scattering intensities of AuNP-only and mixed solutions. The resulting S(q) profile for the mixed solution indicated significant attractive interactions, especially for the smaller AuNPs. Therefore, the sticky sphere model was applied to analyze S(q) to determine the interaction parameters at different Tprep and Tmeas. The attractive interactions between AuNPs were higher at higher Tprep, suggesting that single-chain AC tends to make the interactions between AuNPs more attractive than those for triple-helical AC. Complex formation was also detected by the aggregation-induced surface plasmon absorption shift. More densely packed AuNPs were detected from the absorption spectra for higher AuNP content at which the ζ potential disappeared, while a split absorption band was also found, indicating that not all AuNPs can form a complex with AC at ζ = 0.