Ultrasensitive DNA hybridization biosensor based on polyaniline (PANI) electrochemically deposited onto Pt disc electrode has been fabricated using biotin–avidin as indirect coupling agent to immobilize single-stranded 5′-biotin end-labeled polydeoxycytidine (BdC) probes and 5′-biotin end-labeled 35base-long oligonucleotide probe (BdE) to detect complementary target, using both direct electrochemical oxidation of guanine and redox electroactive indicator methylene blue (MB), respectively. These polyaniline-based disc electrodes have been characterized using differential pulse voltammetry (DPV), Fourier transform infrared spectroscopy (FT-IR), impedance measurements and scanning electron microscopy (SEM) techniques, respectively. Compared to direct electrochemical oxidation of guanine, hybridization detection using MB results in the enhanced detection limit by about 100 times. These DNA immobilized PANI electrodes have hybridization response time of about 60s.