Because of the intrinsic importance of nucleic acids as biotargets, the simple and sensitive detection of nucleic acids is very essential for biological studies and medical diagnostics. In this work, a novel, simple, and selective electrochemical DNA biosensor for the sensitive detection of target DNA (T-DNA) has been developed based on the dual-signaling electrochemical ratiometric method and exonuclease III (Exo III)-assisted target recycling amplification strategy. The assay strategy includes both “signal-on” and “signal-off” elements. The stem-loop (hairpin) DNA capture probe (HP), which was labeled by thiolated methylene blue (MB) at the 3′-protruding termini and ferrocene (Fc) in the middle of the loop, first self-assembled on the gold electrode surface via a Au–S bond. In the presence of T-DNA, the T-DNA hybridized with HP, which triggered the Exo III cleavage process and accompanied the release of T-DNA. As a result, the MB tags were away from and the Fc tags close to the gold electrode surface, leading to the decrease of the oxidation peak current of MB (I MB) and the increase of that of Fc (I Fc). The value of ΔI Fc/|ΔI MB| (ΔI Fc and ΔI MB are the change values of the oxidation peak currents of Fc and MB, respectively) is linear with the concentration of T-DNA from 0.01 pM to 0.8 pM. The detection limit (4.16 fM) of the developed method is much lower than that of the most reported electrochemical DNA biosensors. This strategy provides a simple and sensitive approach for the detection of T-DNA and has promising applications in bioanalysis, disease diagnostics, and clinical biomedicine.