In this work, a simple and ultrasensitive electrochemical DNA biosensor was developed based on the horseradish peroxidase (HRP)-catalyzed electrochemical process and exonuclease III (Exo III)-assisted target recycling amplification strategy. A biotin tag labeled molecular beacon (MB) with hairpin structure was ingeniously designed and assembled on an electrode as a recognition element. The 3' overhang end of the MB labeled with biotin would bind streptavidin (SA)-HRP to give a strong initial signal. Upon target DNA sensing, the MB probe would be stepwise removed by the Exo III accompanied by the release of the target DNA for the successive hybridization and cleavage process. Simultaneously, numerous biotin labeled mononucleotides were liberated, leading to a decrease in the binding of the biotin and SA-HRP polymer on the electrode. As a result, the amplified electrochemical current significantly decreased. Because of the autocatalytic target recycling amplification and the HRP-catalyzed electrochemical process, the dual enzyme-based strategy provided an ultrasensitive approach (down to the 10 fM level) for the electrochemical detection of DNA, and discriminated mismatched DNA from perfectly matched target DNA, which has great potential for early diagnosis in gene-related diseases. (C) 2014 Elsevier Ltd. All rights reserved.