Controlling the reaction kinetics during the nucleation/growth of cupric oxide (CuO) nanostructures is very critical in order to achieve a specific and well-defined morphology. For this purpose, we have slowed down the reaction speed using a mixed solvent concept and successfully obtained a chain-like morphology of CuO nanostructures using hydrothermal method. The CuO chain-like morphology was synthesized using a 1:1 (v/v) ratio of ethylene glycol and water. The morphology and crystalline features of CuO were studied by scanning electron microscopy (SEM) and powder X-ray diffraction techniques. The high resolution transmission electron microscopy revealed 5 nm crystallite size for the CuO material prepared in the mixed solvents. The obtained results have shown that the prepared CuO chains had a monocline phase, containing only Cu and O as main elements as confirmed by energy dispersive spectroscopy. This unique morphology obtained from mixed solvent process has provided a better surface for the loading of urease enzyme, thus it enabled the development of sensitive and selective urea biosensor in phosphate buffer solution of pH 7.4. The physical adsorption method was used to immobilize urease enzyme onto the nano surface of CuO. The fabricated biosensor based on urease/CuO chains has shown a dynamic linear range from 0.0005 to15 mM with a low limit of detection 0.0001 mM. Additionally, a fast response time aroudn1s, h high selectivity, stability, repeatability, storage time, and reproducibility were observed. The effect of pH and temperature on the potentiometric signal of the developed biosensor was also examined. Importantly, the practical aspects of the fabricated urea biosensor were probed and the obtained percent recovery results revealed an outstanding performance. The strategy of using mixed solvent with equal volume ratio would be useful for the preparation of other metal oxides with improved catalytic properties for a wide range of clinical, biomedical and other related applications.