Heavy metals usually coexist with a variety of chelating agents to form heavy metal complexes in industrial wastewater. The decomplexation of heavy metal complexes is the crucial step before the removal of heavy metals via alkaline precipitation process. An efficient synergistic activation of persulfate (PS) with alkali and CuO was used for the simultaneous decomplexation of Cu-ethylenediamine tetraacetic acid (Cu(II)-EDTA) (3.14 mM) and the Cu(II) precipitation. The experimental results demonstrated that nearly complete removal of Cu(II) could be achieved by synergistic activation of PS with alkali and CuO at pH 11 after 2 h of decomplexation reaction. However, sole PS could not effectively decomplex Cu(II)-EDTA (13.5%), while the alkaline activation of PS could accomplish 57.0% removal of Cu(II). Radical scavenger tests indicated that reactive oxygen species (ROS) including SO4•-, •OH and O2•- were responsible for the decomplexation of Cu(II)-EDTA in the synergistic activation of PS with alkali and CuO. As a heterogeneous activator, CuO possessed excellent reusability and long-lasting catalytic activity and the rate constant value (k) of Cu(II) removal showed an increase (from 0.0326 min−1 in the first cycle to 0.0491 min−1 in the 24th cycle) with 24 cycles experiments. Furthermore, the biotoxicity evaluation of treated solution revealed that the biotoxicity of Cu(II)-EDTA contaminated wastewater could be effectively mitigated by the synergistic activation of PS with alkali and CuO because of the efficient precipitation of Cu(II) and oxidative degradation of EDTA organic ligands, which was favorable for the subsequent biochemical treatment.