A simple, signal-off and reusable electrochemical biosensor was developed for sensitive and selective detection of mercury(II) based on thymine-mercury(II)-thymine (T-Hg2+-T) complex and the remarkable difference in the affinity of graphene with double strand DNA (ds-DNA) and single strand DNA (ss-DNA). Our system was composed of ferrocene-tagged probe DNA and graphene. Due to the noncovalent assembly, the ferrocene-tagged probe ss-DNA was immobilized on the surface of graphene nanosheets directly and employed to amplify the electrochemical signal. In the presence of Hg2+, the ferrocene-labeled T-rich DNA probe hybridized with target probe to form ds-DNA via the Hg2+-mediated coordination of T-Hg2+-T base pairs. As a result, the duplex DNA complex kept away from the graphene surface due to the weak affinity of graphene and ds-DNA, and the redox current decreased substantially. Meanwhile, the graphene decorated GCE surface was released for the reusability. Under the optimal conditions, the proposed sensor showed a linear concentration range from 25 pM to 10 mu M with a detection limit of 5 pM for Hg2+ detection. The strategy afforded exquisite selectivity for Hg2+ against other metal ions in real environmental samples. (C) 2015 Elsevier Ltd. All rights reserved.