A label-free DNA hybridization biosensor based on impedance technique was introduced. A 24-mer 5'-mercapto-group ended single-stranded DNA probe (SH-ssDNA) was self-assembled onto Au electrode surface with mercaptoacetic acid (RSH) to form a recognition layer. By Faradic impedance technique, a significant increase of the interfacial electronic transfer resistance Ret was determined when the SH-ssDNA probe/Au electrode hybridized with its complementary sequence. Further, several experimental conditions, including the SH-ssDNA probe immobilization time, hybridization temperature and time, the eletrolyte for impendance detections were optimized. Especially, the proportion of SH-ssDNA and RSH in the immobilization solution was important for a suitable density of ssDNA probe on electrode surface to improve the hybridization efficiency. Based on these optimized conditions, this label-free DNA hybridization biosensor has a low detection limit of 3.0×10-14 mol/L of complementary sequence in solution. When compared with the complementary sequence, the hybridization signal of one-base mismatched sequence and three-base mismatched sequence was 55.6% and 1.3% respectively.