Ochratoxins are a group of mycotoxins produced as secondary metabolites by fungi which contaminate a large variety of food and feed commodities. Due to their teratogenic and carcinogenic properties, ochratoxins present a serious hazard to human and animal health. There is an increasing need to establish a simple sensitive method to detect these toxins. Here we report a rapid and highly sensitive surface plasmon resonance (SPR) assay of ochratoxin A (OTA) using Au nanoparticles for signal enhancement on a mixed self-assembled monolayer (mSAM) surface. A competitive immunoassay format was used for the development of the OTA immunoassay, which is based on the immobilization of target OTA through its ovalbumin (OVA) conjugate with a polyethylene glycol (PEG) linker. The new OTA conjugate (OTA-PEG-OVA) showed remarkably enhanced performance characteristics compared with those based on the immobilization of a commercial bovine serum albumin BSA-OTA conjugate without a PEG linker. Although OTA concentrations as low as 1.5 ng mL(-1) could be directly detected on this surface, the limit of detection (LOD) can be dramatically improved to 0.042 ng mL(-1) for OTA by applying large gold nanoparticles (40 nm) for signal enhancement. Various chemical conditions to minimize the influence of the food matrix on assay performance were also investigated. Grain samples were simply extracted with 50% methanol and liquid samples treated with poly(vinylpyrrolidone) (PVP) (3 or 5%), without any sample clean-up or pre-concentration step prior to analysis. The LODs for OTA in oats and corn were 0.3 and 0.5 ng g(-1), respectively, while in wine and other beverages, LODs ranged from 0.058 to 0.4 ng mL(-1). No cross-reactivity was observed with three other common mycotoxins. In addition, the mSAM/OTA-PEG-OVA surface exhibited high stability with over 600 binding/regeneration cycles. This approach with simple sample preparation provides a powerful tool for the rapid and sensitive quantitative determination of OTA in food matrices.