In this study an electrochemical DNA biosensor with gold nanoparticles (AuNPs) and partially reduced graphene oxide (p-RGO) modified electrode was constructed to detect Listeria monocytogenes My gene sequence. AuNPs were electrodeposited on the surface of carbon ionic liquid electrode (CILE) and then p-RGO was further deposited at controlled electroreduction conditions. On the p-RGO surface unreduced oxygenal groups were present and could covalently interact with the amine group at the 5'-end of probe ssDNA through carbodiimide linkage, and the resulted ssDNA/p-RGO/AuNPs/CILE could be used to hybridize with the target ssDNA sequence. Differential pulse voltammetry was applied to monitor the DNA hybridization with methylene blue as electrochemical indicator. Under the optimal conditions, the electrochemical biosensor could be applied to the detection of target DNA sequence in the concentration range from 1.0 x 10(-13)-1.0 x 10(-6) mol/L with the detection limit of 3.17 x 10(-14) mol/L (3S(0)/S). The sensor exhibited good stability and selectivity to one or three-base mismatched ssDNA sequences. PCR sample of Listeria monocytogenes hly gene sequence was successfully detected, indicating the real application of this biosensor.