The current research performs the novelty fabricated of non-enzymatic biosensor based on copper nanoparticles decorated carbon black nanocomposite modified glassy carbon electrode (Cu/DCB/GCE) by using the microwave method. The prepared nanomaterial was applied for glucose determination in biological fluids and human serum samples. The techniques of SEM, TEM, XPS, XRD, EDS and mapping tests were selected for the morphological, compositions and crystal structure characterizations of the fabricated nanocomposite. In comparison with other electrodes based on metal decorated carbon black, the Cu/DCB/GCE demonstrated the best electrocatalytic property toward glucose oxidation in 0.1 M KOH electrolyte. Cu/DCB/GCE under the optimal potential of +0.5 V in a linear range from 0.5 up to 7000 mu M with a detection limit of 0.1 mu M (S/N=3), the coefficient of correlation R-2=0.999, performed significant high sensitivity of 1595 mu A mM(-1) cm(-2). Rapid amperometric responses of the prepared biosensor in short time with 0.8-2 s were observed by addition of different glucose concentrations in alkaline solution. Modified Cu/DCB/GCE exhibited the best anti-interference property against blood cells and some blood co-existence molecules such as dopamine, uric acid, ascorbic acid, albumin, and globulin. Most importantly, Cu/DCB/GCE biosensor was used for glucose level determination in human blood serum samples. Due to the attractable structure and properties of the fabricated biosensor, it can be suggested for the future development of glucose detection design. (C) 2019 The Electrochemical Society.