Membrane filtration is an efficient technique for rapid and precise separation, yet challenges persist in distinguishing and selectively separating nanoparticles with identical elemental compositions, especially copper-based nanomaterials. Here, we report for the first time the immobilization of thiol-functionalized Fe3O4 nanoparticles (Fe3O4 RSH NPs) onto mixed cellulose ester membranes for the selective separation and preconcentration of copper nanoparticles (Cu NPs). The Fe3O4–RSH NPs–modified membrane demonstrated significantly greater retention of Cu NPs than CuS and CuO NPs of comparable size. The functionalized membrane exhibits exceptional separation performance, achieving over 90% retention of Cu NPs and an enrichment factor of ~23, as verified by inductively coupled plasma mass spectrometry (ICP-MS). Notably, the retention rate of Cu NPs remained unaffected by other copper species, demonstrating the excellent selectivity of the modified membrane toward Cu NPs. Under optimized conditions, the method reached a limit of detection (LOD) of 2.2 pg mL?1 for Cu NPs. Furthermore, the approach enabled successful detection of Cu NPs in river water samples from Chengdu. This strategy provides a rapid, reliable, and cost-effective pretreatment method for trace nanoparticle analysis in environmental samples and offers a promising platform for the development of low-cost, on-site nanoparticle monitoring technologies.