We report on an electrochemical DNA biosensor consisting of a glassy carbon electrode modified with a film of electropolymerized Eriochrome Black T (pEBT) that serves as a functional platform for the immobilization of probe DNA. pEBT was deposited via cyclic voltammetry, and the amino-modified DNA capture probe was covalently linked to the surface via a sulfanilamide coupling reaction. The single step of the assembly process was monitored by atomic force microscopy and electrochemistry. The surface density of DNA probe on the biosensor interface was calculated to be 1.7 × 10−10 mol cm−2 using methylene blue as an electroactive probe. Hybridization experiments showed the peak currents of methylene blue to decrease with increasing concentration of complementary sequence in the range from 5.0 f. to 5.0 pM. The detection limit is as low as 0.11 fM. Selectivity studies showed that the biosensor can discriminate a fully complementary sequence from a single-base mismatch, three-base mismatch, and a fully non-complementary sequence. The biosensor displays good stability and can be regenerated due to the beneficial effects of electropolymerization and covalent immobilization of probe DNA.