In this work, we report a novel electrochemical DNA sensor based on a nonfouling monolayer structure self-assembled at gold surfaces. Self-assembled monolayers (SAMs) with oligo(ethylene glycol) (OEG)-terminated thiols are known to be highly protein-resistant and effectively repel nonspecific adsorption. We found that a mixed SAM structure incorporating thiolated oligonucleotides and OEG thiols (SH-DNA/OEG) exhibited the similar non-fouling feature. More importantly, it allowed facile electron transfer across the monolayer and thus was fully compatible with electrochemical detection. On the basis of this SH-DNA/OEG platform, we developed a sandwich-type electrochemical sensor for the sequence-specific detection of DNA targets. This sensor was able to detect as little as 1 pM target DNA even in the presence of complicated biological fluids such as human serum. We also employed this sensor to directly detect a polymerase chain reaction (PCR) amplicon from the genomic DNA of Escherichia coli K12, which led to a very low detect limit of 60 fg (approximately 10 copies).