Commercially available nanoparticles have been employed as high mass labels for enhancing the binding signals and improving the detection sensitivity of surface plasmon resonance (SPR) assays. Such a signal enhancement is affected by the size and distance of the nanoparticles from the sensing surface. High signal amplifications are expected with increasing nanoparticle size and as the distance between the sensing surface and the nanoparticle is decreased. This paper describes a new way to improve the SPR assay sensitivity of small molecules using a mixed self-assembled monolayer (mSAM) surface to bring the nanogold particles close to the sensing surface. Progesterone (P4) was conjugated to ovalbumin (OVA) with an oligoethylene glycol (OEG) linker to form protein conjugate (P(4)-OEG-OVA), which was immobilized onto the mSAM surface. Inhibition immunoassays based on this mSAM/P4-OEG-OVA surface have demonstrated that 10nm nanogold dramatically improved the assay sensitivity of progesterone, lowering its limit of detection (LOD) from the original 372.7 to 4.9 ng L(-1). In addition, the high stability of the mSAM/P4-OEG-OVA surface was demonstrated by the use of a single chip for over 400 binding/regeneration cycles without any significant drop in antibody binding capacity and baseline shift.