A biocompatible nanostructured poly(xanthurenic acid)–Fe2O3/reduced graphene oxide (PXa–Fe2O3/RGO) platform was constructed for electrochemical sensing of DNA hybridization. The affinity of nanosized Fe2O3 with xanthurenic acid (Xa) monomer coupled with the π–π* stacking and hydrogen bonding between the conjugated graphene oxide layers and aromatic ring of Xa enhanced the electropolymerization efficient, resulting in an improved electrochemical response of PXa. The obtained PXa–Fe2O3/RGO nanocomposite integrated the advantages of the Fe2O3/RGO and PXa, owning rich-conjugated structures, fine biocompatibility, and good conductivity. The immobilization of the probe DNA was successfully achieved via noncovalent bonding due to π–π* interaction between the conjugated PXa–Fe2O3/RGO and DNA bases. After the hybridization of probe DNA with target DNA, the formation of double-helix structure induced dsDNA to release from the surface of conjugated nanocomposite, accompanied with the self-signal regeneration of nanocomposite (“signal-on”). The self-signal changes induced by DNA immobilization and hybridization could be sensitively recognized via cyclic voltammetry and electrochemical impedance spectroscopy, and the synergistic effect of the graphene-based nanocomposite improved the sensitivity for the target DNA detection.