In this work, three different spherulitic nanostructures Cu–CuO
A
, Cu–CuO
B
, and Cu–CuO
C
were synthesized in water-in-oil microemulsions by varying the surfactant concentration (30 mM, 40 mM, and 50 mM, respectively). The structural and morphological characteristics of the Cu–CuO nanostructures were investigated by ultraviolet–visible (UV–vis) spectroscopy, X-ray diffraction, scanning electron microscopy, and high-resolution transmission electron microscopy techniques. The synthesized nanostructures were deposited on multiwalled carbon nanotube (MWCNT)-modified indium tin oxide (ITO) electrodes to fabricate a nonenzymatic highly sensitive amperometric glucose sensor. The performance of the ITO/MWCNT/Cu–CuO electrodes in the glucose assay was examined by cyclic voltammetry and chronoamperometric studies. The sensitivity of the sensor varied with the spherulite type; Cu–CuO
A
, Cu–CuO
B
, and Cu–CuO
C
exhibited a sensitivity of 1,229, 3,012, and 3,642 µA mM
−1
·cm
−2
, respectively. Moreover, the linear range is dependent on the structure types: 0.023–0.29 mM, 0.07–0.8 mM, and 0.023–0.34 mM for Cu–CuO
A
, Cu–CuO
B
, and Cu–CuO
C
, respectively. An excellent response time of 3 seconds and a low detection limit of 2 µM were observed for Cu–CuO
B
at an applied potential of +0.34 V. In addition, this electrode was found to be resistant to interference by common interfering agents such as urea, cystamine, L-ascorbic acid, and creatinine. The high performance of the Cu–CuO spherulites with nanowire-to-nanorod outgrowths was primarily due to the high surface area and stability, and good three-dimensional structure. Furthermore, the ITO/MWCNT/Cu–CuO
B
electrode applied to real urine and serum sample showed satisfactory performance.