Copper ion (Cu (II)) pollution has attracted much attention due to its remarkable toxic domino effect at excess amount. Efficient Cu (II) ions removal is thus a prerequisite for wastewater recycling. Herein, we present a facile and environmentally benign strategy to fabricate thiol (SH)-functionalized Fe3O4@C nanoparticles (denoted as Fe3O4@C-SH NPs) based on one-step self-assembling of a bifunctional oligopeptide with a sequence of Cys-Lys-Cys-Lys-Cys-Lys (CK–VI) for highly efficient removal of copper ions (Cu (II)) in aqueous solutions. Under the physiological conditions, CK–VI readily self-organized into a robust and tailor-made functional monolayer predominately composed of well-packed β-sheets on the surface of Fe3O4@C NPs with their thiol groups standing on the outermost layer. The resulting Fe3O4@C-SH NPs containing abundant thiol active sites exhibited excellent adsorption capacity (up to 28.8 mg g−1) and selectivity for Cu (II) ions over coexisting ions. Compared with other covalent grafting methods with multistep processes and in harsh conditions, the proposed oligopeptides assembly-based coating method makes it possible to rapidly fabricate the Fe3O4@C-SH NPs in a simple mild one-step aqueous process with low cost. The current study provides facile and environmentally friendly approaches to rapidly tailor multifunctional surfaces of NPs for various toxic metal ions removal from wastewater.