In this work, a signal-off double probes electrochemical DNA sensor was developed to simultaneously detect Legionella and Legionella pneumophila. We designed hairpin probes which modified by ferrocene/methylene blue (Fc/MB) for selective electrochemical detecting Legionella and Legionella pneumophila. The hairpin probes were immobilized by Au-S chemical bond reaction on the surface of AuE electrodes, which can be cleaved by EcoRI in the absence of Legionella, but cannot be cleaved in the presence of Legionella or Legionella pneumophila. The current changes before and after DNA hybridization and enzymatic cleavage were monitored by the proposed DNA sensor With differential pulse voltammetry. Electrochemical signals was generated by Fc/MB. There was a good linear relationship between the peak current value and the logarithmic concentration of Legionella or Legionella pneumophila. The linear regression equation Of Legionella was 1(nA) = 111.57logC(mol/L) + 1084.0, R-2 = 0.9902; and the linear regression equation of Legionella pneumophila was I(nA) = 126.09logC(mol/L) + 1238.2, R-2 = 0.9945. The linear range had been estimated from 2.0 x 10(-9) to 2.0 x 10(-4)mol/L. Legionella, Legionella pneumophila and other batteria were sensitively distinguished by the proposed DNA sensor. And the sensor could be used for detecting the Legionella pneumophila samples from patients or environments. (C) 2017 Elsevier B.V. All rights reserved.