In this study, a novel electerochemical sensor was made for determination of glucose and acyclovir based on three dimensional reduced graphene (3D-rGO)/multi wall carbon nanotube (MWCNT) nanocomposite modified with nickel and copper oxide nanoparticles (NiOCu 2 O). The different ratio of Ni and Cu were investigated and the optimum amount selected for determination. NiOCu 2 O were assembled on the sheets of rGO and MWCNT are interconnected between the rGo sheets and appeared the 3D network of rGO/MWCNT. The final nanocomposite was named NiOCu 2 O@3D-rGO/MWCNT and characterized by scanning electron microscopy (SEM), transition electron microscopy (TEM), energy dispersive spectroscopy (EDS), atomic force microscopy (AFM), X-ray diffraction spectroscopy (XRD) and FT-IR spectroscopy. The NiOCu 2 O@3D-rGO/MWCNT has a porous structure with uniform dispersion of NiOCu 2 O. In the first section of this study, amperometric sensor (NiOCu 2 O@3D-rGO/MWCNT/GCE) was made for determination of glucose. The activity of electrocatalyst toward electrooxidation of glucose was checked at the optimized condition by cyclic voltammetry and amperomtery technique in 0.15 mol L –1 NaOH. The prepared sensor gives a detection limit of (0.04 µmol/L) a dynamic range of (0.10 – 9.00 ×10 2 µmol/L ) and high stability for 3000 s. In the second section of this study, electerochemical sensor (NiOCu 2 O@3D-rGO/MWCNT/GCE) was made for determination of acyclovir. The nanocomposite film showed excellent electrocatalytic activity towards the oxidation of acyclovir in universal buffer (pH= 5.0). The electrocatalytic activity of the modified electrode towards acyclovir was systematically studied by cyclic voltammetry and differential pulse voltammetry techniques. Under the optimum conditions, the linear ranges were (1.00 × 10 ? 2 – 1.00 µmol/L) and (1.00 – 1.00× 10 2 µmol/L) with limits of detection (LOD, S/N = 3) of (0.006 µmol/L). Furthermore, the modified electrode was applied to real sample analysis.