In this study, a highly sensitive biosensor based on a 3D nano-structured sensing electrode made from an anodic aluminum oxide (AAO) barrier layer with the uniform distribution of gold nanoparticles (GNPs) is proposed for label-free detection of orchid viruses. This nanostructured electrode was utilized for developing a label-free sensing system based on electrochemical impedance spectroscopy (EIS) to directly detect the Odontoglossum ringspot virus (ORSV) concentration from orchid leaves. The ORSV antibody (anti-ORSV) was employed as a detecting probe and was covalently immobilized on the nanostructured sensing electrode surface through a self-assembled monolayer (SAM) method. Through a selective binding interaction between specific antigen and antibody pairs, our nanostructured biosensors can provide a high specificity and accuracy sensing result. Our experimental results revealed that the specific binding of ORSV onto the immobilized anti-ORSV gave rise to the incremental interfacial charge transfer resistance of the electrode, allowing one to distinguish between healthy and infected orchids in a fast and accurate manner. The linear detection range is 50,000 ng/mL-0.5 ng/mL with R-2 = 0.9686. A detection limit of 0.345 ng/mL can be achieved. These results demonstrated that our ORSV biosensor is feasible for field applications. (C) 2018 The Electrochemical Society.