The authors describe a voltammetric method for the detection of the nuc ssDNA sequence originating from Staphylococcus aureus by using a carbon ionic liquid electrode modified with electrodeposited three-dimensional graphene (3DGR). Probe ssDNA was electrostatically adsorbed on the modified electrode by a potentiostatic method. The porous structure and large surface area of 3DGR greatly increase the amount of immobilized probe ssDNA on the interface, which is beneficial for the reaction with target ssDNA. By using Methylene Blue (MB) as the electrochemical probe, the reduction peak current of MB (best measured at -0.30V vs. SCE) can be used for detecting hybridization. The differential pulse voltammetric current of MB increases linearly in the 1.0 × 10-12mol L-1 to 1.0 × 10-6mol L-1 nuc concentration range, and the detection limit is 3.3× 10-13mol L-1 (at 3σ). The DNA sensor was successfully applied to the determination of the PCR product of the gene in pork. Graphical abstract Response of an electrochemical DNA biosensor based on the use of a carbon ionic liquid electrode modified with three-dimensional graphene. It enables sensitive voltammetric detection of the specific sequence of the Staphylococcus aureus nuc gene.