Endotoxin is a highly toxic stimulator originated from the outer membrane of Gram-negative bacteria, which should be monitored sensitively and selectively for human health concerns. Traditional detection methods mainly rely on limulus amoebocyte lysate assay. However, it suffers drawbacks like the narrow detection range, and the results may be environment-dependent. In this work, we have developed a sensitive electrochemical biosensor for endotoxin assay. Peptide is first designed as specific recognition element toward endotoxin. Graphene oxide and DNA-modified gold nanoparticles are then used to enhance the electrochemical signal. The analytical performances are excellent with the limit of detection as low as 0.001 EU mL-1. This method has also been successfully applied in endotoxin assay in complex biological samples, which may have great potential use.