Novel nickel and copper nanoparticles decorated multi-walled carbon nanotubes (Ni/Cu/MWCNT) have been successfully fabricated for sensitive nonenzymatic glucose detection by the sequential electro-deposition of nickel and copper nanoparticles (NPs) on an MWCNT-modified electrode. X-ray diffraction (XRD) and atomic force microscopy (AFM) analyses reveal that the Ni and Cu NPs were successfully deposited on the MWCNTs in this hybrid composite. The electrode shows good activity towards glucose oxidation with low over-potential and a current response that is 2.5–20 times greater than that obtained using Ni/GCE, Cu/GCE, Ni/Cu/GCE, Ni/MWCNT/GCE, and Cu/MWCNT/GCE. The optimised conditions based on current response are a Ni:Cu ratio of 1:1 and pH 13. Amperometry (Eapp. = +0.575 V) indicates a short response time of 1 s; two specific linear ranges of 2.5 × 10−8–8 × 10−4 M and 2 × 10−3–8 × 10−3 M, with high sensitivities of 2633 μA mM−1 cm−2 and 2437 μA mM−1 cm−2, respectively; and a low detection limit of 2.5 × 10−8 M (S/N = 3). This electrode can effectively analyse glucose concentration in human serum samples, avoiding interference, and is a promising nonenzymatic glucose sensor due to its low overpotential, high sensitivity, good selectivity, good stability, fast response, and low cost.