Sensitive and selective detection of proto-oncogenes, especially recognition of point mutation, is of great importance in cancer diagnosis. Here, a ligation-mediated technique is demonstrated for the construction of an intertwined three-dimensional DNA nanosheet (3D SDN) on an electrode surface from only two palindromic hairpin probes (HP1 and HP2), creating a powerful electrochemical biosensor (E-biosensor) for the detection of the p53 gene. First, a capturing probe (CP) is immobilized on an electrode surface via Au-S chemistry, forming an electrochemical sensing interface. In the presence of the target p53 (T), the triggering probe is covalently linked to CP by a ligase. Moreover, target hybridization/ligation/dehybridization process is repeated, amplifying the target hybridization event and increasing the content of surface-confined triggering fragments. As a result, HP1 is opened and in turn interacts with HP2, forming intertwined 3D SDN where HP1 and HP2 are alternately arranged in parallel. Common hybridization and interaction between palindromic fragments are responsible for the assembly in the horizontal and vertical directions, respectively. An electrochemical indicator, methylene blue (MB), can be inserted into 3D SDN, generating a strong electrochemical signal. Utilizing the 3D SDN-based E-biosensor, the target DNA is detected down to 3 fM with a linear response range from 10 fM to 10 nM. Single point mutations are reliably identified even in fetal bovine serum and cellular homogenate. Because of the several advantages of simple design, good universality, inexpensive instrumentation, high assay specificity, and sensitivity, the 3D SDN-based E-biosensor is expected to provide a potential platform for screening point mutation required by early clinical diagnostics and medical research.