We reported a single-use non-enzymatic glucose biosensor based on a printed CuO nanoparticles film on thin gold film electrode. This three-electrode configuration biosensor consisted of a thin gold film (10 nm) working and counter electrodes and a thick-film printed Ag/AgCI reference electrode. The biosensor was fabricated on a polyethylene terephthalate (PET) substrate. CuO nanoparticles with an average particle size of similar to 7 nm were synthesized by a one-step precipitation approach, formulating an aqueous-based nanoparticles ink. A CuO nanoparticles square area of 0.9 x 0.9 mm(2) was printed on thin film Au working electrode (phi)=1.4 mm) of the biosensor by micro-plotter technology. The remarkable resolution and dimensional accuracy of micro-plotter printing technology resulted in a better development of nano-catalyst biosensors compared to conventional ink-jet printing or other techniques. X-ray Photoelectron Spectroscopy (XPS) and Transmission Electron Microscopy (TEM) were used to characterize the printed biosensor. Field Emission Scanning Electron Microscopy (FESEM) characterizations of the copper oxide printed features showed a nano-porous morphology together with high printing resolution. Cyclic Voltammetry (CV) and chtonoamperometry (CA) measurements revealed the considerable electrocatalytic performance of the printed biosensor having high sensitivity (2419.8 mu A mM(-1) cm(-2)), acceptable stability and excellent interference rejection against ascorbic acid (AA), uric acid (UA), dopamine (DA), lactose, mannose and maltose. Interference rejection studies were performed in undiluted human serum to verify the functionality of the sensor for real samples. The developed biosensor showed a linear response toward glucose in the range of 0.1 to 6.5 mM. The lower detection limit of the biosensor was measured as 0.5 mu M glucose. This study suggested a high performance robust glucose biosensor not only suitable for biomedical single-use in vitro application but also for long-term glucose monitoring in industrial processes. (C) 2017 Elsevier B.V. All rights reserved.