Abstract: The surface of glassy carbon electrode (GCE) was first activated by electrochemical technique, and then immobilized with legionella′s specific gene sequence probe ssDNA, which has been modified with aminogroup by coupling activator. Thus, the electrochemical biosensor for determination of the leginonella′s DNA segment was fabricated and its properties were characterized and optimized by using electrochemical AC impedance technique. As shown by the experimental results, the complementary legionella′s DNA segments were effectively recognized by the change of impedance signals given by the biosensor before and after hybridization. Under optimum conditions, linear relationship between the values of increase in impedance value (ΔR) and the concentration of the DNA target standard strand was obtained in the range of 5.0×10-9-2.5×10-8mol·L-1, with value of detection limit (3S/N) of 1.0×10-9mol·L-1.