Cu2+ has increasingly become a great threat to the natural environment and human health due to its abundant content and wide application in various industries. DL-Mercaptosuccinic acid and ZnS-modified Fe3O4 nanocomposites were designed, synthesized, and applied in the determination of Cu2+. The prepared nanocomposites were characterized by scanning electron microscopy (SEM), transmission electron microscopes (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), X-ray photoelectron spectroscopy (XPS), vibrating sample magnetometer (VSM), and thermogravimetric analyzer (TG). The magnetic fluorescent nanoprobe exhibited highly selective and sensitive fluorescence-quenching characteristics with Cu2+ ions. The fluorescence detection linear range was 0–400 μM, with the detection limit being 0.489 μM. In addition, the magnetic fluorescent nanoprobe exhibited a high adsorption and removal rate for Cu2+. It had been successfully applied to detect Cu2+ in real water samples with a satisfactory recovery rate. The magnetic fluorescent nanoprobe could simultaneously realize the functions of enrichment, quantitative detection, and separation, reduce the pollution of copper ions and probes, and establish an environment-friendly detection method. Consequently, the magnetic fluorescent nanoprobe offered a new pathway for the removal and detection of not only Cu2+ but also other heavy metal ions in water.