In this work, a very simple, label-free, isothermal, and ultrasensitive electrochemical DNA biosensor has been developed on the basis of an autocatalytic and exonuclease III (Exo III)-assisted target recycling amplification strategy. A duplex DNA probe constructed by the hybridization of a quadruplex-forming oligomer with a molecular beacon is ingeniously designed and assembled on the electrode as recognition element. Upon sensing of the analyte nucleic acid, the strand of molecular beacon in the duplex DNA probe could be stepwise removed by Exo III accompanied by the releasing of target DNA and autonomous generation of new secondary target DNA fragment for the successive hybridization and cleavage process. Simultaneously, numerous quadruplex-forming oligomers are liberated and folded into G-quadruplex-hemin complexes with the help of K(+) and hemin on the electrode surface to give a remarkable electrochemical response. Because of this autocatalytic target recycling amplification and the specifically catalyzed formation of G-quadruplex-hemin complexes, this newly designed protocol provides an ultrasensitive electrochemical detection of DNA down to the 10 fM level, can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases. It further could be developed as a universal protocol for the detection of various DNA sequences and may be extended for the detection of aptamer-binding molecules.