In this work, a sensitive electrochemical DNA sensor based on an avidin modified electrode and a DNAfunctionalized Au nanoparticle (DFNP) was developed. The DNA-functionalized Au nanoparticle contained two kinds of DNA, one is hairpin probe DNA with a biotin at the 30 terminal and a thiol at the 50 terminal, the other is methylene blue (MB)-labeled linear signal DNA. Without hybridizing with the target DNA, the loop of the hairpin impeded biotin linked with avidin on electrode. However, after target hybridization, the hairpin was opened and biotin was recognized by avidin which resulted in a DNAfunctionalized Au nanoparticle brought on the electrode surface. Electrochemical signals of MB bound to signal DNA were measured by differential pulse voltammetry (DPV). Taking advantage of amplification effects of the AuNP and binding specificity of the hairpin probe, this DNA biosensor greatly simplified the electrochemical DNA detection method and displayed high specificity in DNA detection. By using this newmethod, we demonstrate that this prototype sensor has been able todetect as lowas picomolar DNA targets with excellent differentiation ability even for a single mismatch.