Presented herein is a detailed study into the controlled adsorption of polyethylenimine (PEI) onto 50 nm crystalline magnetite nanoparticles (Fe3O4 NPs) and how these PEI-coated Fe3O4 NPs can be used for the magnetic capture and quantification of ultratrace levels of free cupric ions. We show the ability to systematically control the amount of PEI adsorbed onto the Fe3O4 magnetic nanoparticle surfaces by varying the concentration of polymer during the adsorption process. This in turn allows for the tailoring of important colloidal properties such as the electrophoretic mobility and aggregation stability. Copper adsorption tests were carried out to investigate the effectiveness of PEI-coated Fe3O4 NPs in copper remediation and detection. The study demonstrated that the NPs ability to bind with copper is highly dependent on the amount of PEI adsorbed on the NP surface. It was found that PEI-coated Fe3O4 NPs were able to capture trace levels (similar to 2 ppb) of free cupric ions and concentrate the ions to allow for detection via ICP-OES. More importantly, it was found that due to the amine-rich structure of PEI, the PEI-coated Fe3O4 NPs selectively adsorb toxic free cupric ions but not the less toxic EDTA complexed copper. This unique property makes PEI-coated Fe3O4 NPs a novel solution for the challenge of separating and quantifying toxic cupric ions as opposed to the total copper concentration of a sample.