Display omitted ► Gold and graphene modified carbon ionic liquid electrode was fabricated. ► ssDNA probe was covalently-linked on the electrode to prepare the DNA biosensor. ► Polymerase chain reaction product of Listeria monocytogenes gene was detected. A new electrochemical DNA biosensor was fabricated by using a dendritic gold nanoparticles and electrochemical reduced graphene (GR) composite modified carbon ionic liquid electrode (CILE) as the platform. Ionic liquid 1-butylpyridinium hexafluorophosphate was used as the binder for the preparation of CILE and GR film was further decorated on the CILE surface by electrochemical reduction. Then the dendritic nanogold was electrodeposited on the surface of GR/CILE to get a modified electrode as Au/GR/CILE, which was further used for the formation of mercaptoacetic acid self-assembling film. The amino modified ssDNA probe sequence was covalently linked with mercaptoacetic acid to get the ssDNA modified electrode for the further hybridization. Methylene blue (MB) was used as the electrochemical indicator for monitoring the hybridization reaction after hybridized with the target ssDNA. Under the optimal conditions the specific Listeria monocytogenes hly ssDNA sequences could be detected by measuring the differential pulse voltammetric responses of the accumulated MB molecules on dsDNA molecules. The linear concentration range was achieved from 1.0×10−12 to 1.0×10−6mol/L with the detection limit as 2.9×10−13mol/L (3σ). This electrochemical DNA sensor exhibited excellent selectivity with the good discrimination ability of one-base and three-base mismatched ssDNA sequences. The polymerase chain reaction product of L. monocytogenes hly gene that extracted from deteriorated fish was successfully detected, which indicated that this electrochemical DNA sensor could be further used for the detection of specific ssDNA sequence in real biological samples.