In this work, we fabricated a sensitive electrochemical DNA biosensor for the detection of target DNA. Aminobenzoic acid (ABA) was firstly electropolymerized on the surface of the glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes with carboxyl groups (MWCNTs) by cyclic voltammetry (CV). Gold nanoparticles (AuNPs) were subsequently introduced to the surface of PABA–MWNTs composite film by electrochemical deposition mode. Probe DNA was immobilized on the surface of AuNPs through Au–S bond. Scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) were used to investigate the film assembly process. Differential pulse voltammetry (DPV) was used to monitor DNA hybridization event by measurement of the intercalated adriamycin. Under the optimal conditions, the increase of reduction peak current of adriamycin was linear with the logarithm of the concentration of the complementary oligonucleotides from 1.0 × 10−12 to 5.0 × 10−9 M with a detection limit of 3.5 × 10−13 M. This DNA biosensor has a good stability and reproducibility.