ZrO2 microparticles and self-doped polyaniline nanofibres (nanoSPAN, copolymer of aniline and m-aminobenzenesulfonic acid) were utilized to fabricate synergistic membranes for the DNA hybridization sensing. The nanoSPAN was coated on the surface of glassy carbon electrode to form a negatively charged nanostructure monolayer. Then ZrO2 microparticles with strong affinity toward the oxygen-containing groups were electrochemically deposited on nanoSPAN by using cyclic voltammetry, which acted as a bridge to link the –SO3H group of the conducting polymer nanostructures and the phosphate group of the DNA. The architecture of the as-prepared ZrO2/nanoSPAN, the immobilization and hybridization of DNA were characterized by scanning electron microscopy, electrochemical impedance spectroscopy and cyclic voltammetric technology, respectively. The charge transfer resistance observed for the Fe ( CN ) 6 3 - / 4 - redox marker was related to the adhered ssDNA and the hybridized dsDNA, which can be used as the highly sensitive label-free impedance sensing for the sequence-specific DNA detection. The transgenic fragment of phosphoenolpyruvate carboxylase (PEPCase) gene in transgenic rape was successfully detected by using this DNA electrochemical biosensor. The linear range was from 1.0×10−12 M to 1.0×10−6 M with a detection limit of 3.4×10−13 M. This is the first application of nanoSPAN combined with ZrO2 microparticles to fabricate a DNA electrochemical biosensor with a synergistic performance for the sensitive and rapid detection of DNA hybridization.