A sensitive electrochemical DNA biosensor was prepared based on mercaptoacetic acid (MAA)/gold nanoparticles (AuNPs) modified electrode. Probe DNA (NH2-DNA) was covalently linked to the carboxyl group of MAA in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxyl-succinimide (NHS). Scanning electron microscopy (SEM) and electrochemical impedance spectra (EIS) were used to investigate the film assembly process. The DNA hybridization events were monitored by differential pulse voltammetry (DPV), and adriamycin was used as the electrochemical indicator. Also the factors influencing the performance of the DNA hybridization were investigated in detail. Under the optimal conditions, the signal was linearly changed with target DNA concentration increased from 5.0 × 10−13to 1.0 × 10−9 M and had a detection limit of 1.7 × 10−13 M (signal/noise ratio of 3). In addition, the DNA biosensor showed good reproducibility and stability during DNA assay.