Electrodes modified with organic layers and nanoparticles are promising tools for analytical devices. A major challenge however is how to make electrodes that resist the nonspecific protein adsorption from biological media? Herein, we report gold electrode-organic layer-gold nanoparticle constructs as a platform for electrochemical biosensing. This study focuses on the fabrication of antifouling electrodes with the organic layers formed by two different approaches; self-assembled monolayer (SAM) procedure and electrochemical reduction of aryl diazonium salts, followed by the attachment of gold nanoparticle (AuNP) to allow the electrochemistry to proceed. The results show that the electrodes modified with ethylene oxide-based SAM enable a better antifouling behaviour with less adsorption of human serum albumin (HSA) as compared to the electrodes modified with ethylene oxide-based aryl diazonium-derived layer. Furthermore, findings suggest such distinction could be due to the surface packing and/or conformation of organic film on Au electrodes. (C) 2016 Elsevier B.V. All rights reserved.