A novel and sensitive electrochemical DNA biosensor based on porous pseudo-carbon paste electrode (PPCPE) for DNA immobilization and enhanced hybridization detection is described. PPCPE was fabricated by mixing polymethyl methacrylate (PMMA) microspheres as the template, graphite powders as the filler, pyrrole as the precursor of polymer which actually acted as the paste. After the polymerization of pyrrole catalyzed by Fe3+, the template PMMA microspheres were removed to form PPCPE. The pore size were determined by SEM observations, their diameters were in the range from 2 to 5 microm, and the specific surface area of PPCPE was 42.76 m2/g by N2 adsorption at 77 K using the Brunauer Emmet Teller (BET) method. The hybridization reaction on the electrode was monitored by anodic stripping voltammetry (ASV) analysis using the method of gold nanoparticle-catalyzed silver enhancement. Compared with previous DNA sensors the attached DNA and complementary DNA detection sensitivity were dramatically increased. The experiments of ASV showed that the peak current of Ag at PPCPE were linear with the amount of complementary oligonucleotide ranging from 1 to 80 nM and 0.08 to 1 nM, the detection limit was as low as 0.05 nM.