DNAs are one of the most fundamental molecules for life. Quantification of specific sequences is of great importance for biological research and clinical diagnosis. In order to determine extremely low abundant DNAs, we herein develop a novel electrochemical genosensor taking advantage of a smart bipedal DNA walking machine. Magnetic nanomaterials are first employed to enrich target DNA. Strand displacement amplification initiated by target DNA is then designed on the surface of the nanomaterials, the products of which can be used to trigger bipedal DNA walking on the surface of an electrode. Benefiting from triple amplification, ultrahigh sensitivity is achieved for electrochemical analysis of DNA. More importantly, the proposed strategy opens a new avenue for employing the bipedal DNA walker for sensitive detection of various biomolecules with signal amplification.