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An electrochemical sensor for the detection of the natural double-stranded DNA (dsDNA) damage induced by PbSe quantum dots (QDs) under UV irradiation was developed. The biosensing membranes were prepared by successively assembling 3-mercaptopropionic acid, polycationic poly (diallyldimethyl ammonium) and dsDNA on the surface of the gold electrode. Damage of dsDNA was fulfilled by immersing the sensing membrane electrode in PbSe QDs suspension and illuminating it with an UV lamp. Cyclic voltammetry was utilized to detect dsDNA damage with Co(phen)3+3 as the electroactive probe. The UV irradiation, Pb2+ ions liberated from the PbSe QDs under the UV irradiation and the reactive oxygen species (ROS) generated in the presence of the PbSe QDs also under the UV irradiation were the three factors of inducing the dsDNA damage. The synergistic effect of the three factors might dramatically enhance the damage of dsDNA. This electrochemical sensor provided a simple method for detecting DNA damage, and may be used for investigating the DNA damage induced by other QDs.