Colloidal Au-amplified surface plasmon resonance (SPR), like traditional SPR, is typically used to detect binding events on a thin noble metal film. The two major concerns in developing colloidal Au-amplified SPR lie in 1) the instability, manifested as a change in morphology following immersion in organic solvents and aqueous solutions, and 2) the uncontrollable interparticle distance, determining probe spacing and inducing steric hindrance between neighboring probe molecules. This may introduce uncertainties into such detecting techniques, degrade the sensitivity, and become the barricade hampering colloidal Au-based transducers from applications in sensing. In this paper, colloidal Au-amplified SPR transducers are produced by using ultrathin Au/Al2O3 nanocomposite films via a radio frequency magnetron co-sputtering method. Deposited Au/Al2O3 nanocomposite films exhibit superior stability, and average interparticle distances between Au nanoparticles with similar average sizes can be tuned by changing surface coverage. These characteristics are ascribed to the spacer function and rim confinement of dielectric Al2O3 and highlight their advantages for application in optimal nanoparticle-amplified SPR, especially when the probe size is smaller than the target molecule size. This importance is demonstrated here for the binding of protein (streptavidin) targets to the probe (biotin) surface. In this case, the dielectric matrix Al2O3 is a main contributor, behaving as a spacer, tuning the concentration of Au nanoparticles, and manipulating the average interparticle distance, and thus guaranteeing an appropriate number of biotin molecules and expected near-field coupling to obtain optimal sensing performance.