In this work, a copper oxide nanoparticles (CuO-NPs) electro-catalyst was prepared on three-dimensional graphene (3DGR) and used as non-enzymatic sensing platform for malathion detection. The structural and morphological properties of the CuO-NPs/3DGR nanocomposite were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). 3DGR framework with pleated structures provided a large surface area for high dispersion of the CuO-NPs. The obtained CuO-NPs/3DGR nanocomposite was noted to possess high affinity towards malathion, and thus the redox reaction of CuO could be hindered. This feature was utilized to design as a simple strategy for electrochemical detection of malathion. The experimental parameters including accumulation time, pH value of buffer solution, and the volume of modified composite were optimized. Under optimal conditions, the CuO-NPs/3DGR modified electrode could detect malathion down to 0.01 nM on the basis of the suppressed signal measurements. The high sensitivity can be ascribed to the fact that the 3DGR worked as high-surface-area substrate to anchor CuO-NPs and provided excellent conductive pathways to promote electron transfer. Furthermore, for the determination of malathion in real samples, the sensor showed good recovery, stability, and selectivity.