In the present work, we describe a simple electrochemical synthesis of CuO nanoflakes (CuO-NFs) using Cu-melamine complex. The as-prepared CuO nanoflakes was characterized by different physicochemical methods such as high-resolution scanning electron microscopy, elemental analysis and elemental mapping. The effect of different potential cycling towards the morphology of CuO-NFs was studied and discussed. Furthermore, CuO-NFs modified electrode was used as an electrocatalyst for oxidation of glucose in 0.1 M NaOH, and the observed electrochemical oxidation current of glucose was higher than CuNPs modified electrode. Amperometric i-t method was used for the determination of glucose using CuO-NFs modified electrode. Under optimal conditions, the amperometric i-t response of the sensor was linear over the glucose concentrations ranging from 1.0 µM to 1.445 mM with the detection limit of 0.35 µM. In addition, the selectivity of the sensor was tested in the presence of different potentially interfering compounds. The practicality of the sensor was also evaluated in human serum samples and shows acceptable recovery of glucose