To address the requirement of ultra-sensitive detection of trace mercury(II) (Hg2+) ions in the environment and food, we developed an electrochemical biosensor with super-sensitivity, extremely high selectivity, and reusability. This biosensor comprised two signal amplification components: a three-dimensional macroporous dealloyed (3DOMD) Au-Ag thin-film electrode and a multifunctional encoded Au@Pt nanocage (APNC). As a platform for immobilized capture DNA (cDNA), a 3DOMD Au-Ag thin film prepared by a dealloying method with an active surface area 4.8 times higher than that of 3D macroporous gold films generated by cyclic voltammetry (CV) with sulfuric acid was capable of increasing the sensing surface area while also strengthening the electron transport capacity of the sensing substrate due to its multilayered multi-porous framework. In the presence of Hg2+, probe DNA (pDNA) could be hybridized with the mismatched capture DNA (cDNA) through stable thymine-Hg2+-thymine (T-Hg2+-T) linkages, connecting thionine-APNC to the electrode surface and utilizing the large specific surface area to accomplish highly sensitive detection of Hg2+. With an extremely low Hg2+ detection limit of 2 pM and a detection range from 0.01 to 1000 nM, this technique opened up a new avenue for the ultrasensitive detection of a wider range of heavy metal ions or biomolecules.