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Abstract: A novel and highly sensitive electrochemiluminescence (ECL) assay based on magnetic beads separation/collection process and thymine–Hg2+–thymine (T–Hg2+–T) coordination chemistry has been designed to detect Hg2+ ions in aqueous solution. In this protocol, two amine-terminated complementary DNA probes with five thymine–thymine (T–T) mismatches were introduced. One was coupled with carboxyl-modified magnetic beads and the other was labeled with tris (2, 2-bipyridine) ruthenium (II) (TBR). The couple of DNA probes, in the presence of Hg2+, can form double-stranded structure via the Hg2+-mediated coordination of T–Hg2+–T base pair. Therefore, they can be collected on the surface of electrode using a magnetic field. On the electrode, TBR labels and tripropylamine (TPA) could react to emit photons, which were detected by a custom-built ECL system. The detection limit for Hg2+ in this assay is 5nM, which is below the upper limit of Hg2+ for drinkable water mandated by United States Environmental Protection Agency (EPA), 10nM. To the best of our knowledge, this is the first example of ECL assay applying to detect Hg2+ with highly sensitivity and selectivity. [ABSTRACT FROM AUTHOR]