As an important and necessary step of sampling biological specimens, the separation of malignant cells from a mixed population of cells usually requires sophisticated instruments and/or expensive reagents. For health care in the developing regions, there is a need for an inexpensive sampling method to capture tumor cells for rapid and accurate diagnosis. Here we show that an underexplored generic difference-overexpression of ectophosphatases-between cancer and normal cells triggers the d-tyrosine phosphate decorated magnetic nanoparticles (Fe3O4-p(d-Tyr)) to adhere selectively on cancer cells upon catalytic dephosphorylation, which enables magnetic separation of cancer cells from mixed population of cells (e.g., cocultured cancer cell (HeLa-GFP) and stromal cells (HS-5)). Moreover, the Fe3O4-p(d-Tyr) nanoparticles also selectively inhibit cancer cells in the coculture. As a general method to broadly target cancer cells without highly specific ligand-receptor interactions (e.g., antibodies), the use of an enzymatic reaction to spatiotemporally modulate the state of various nanostructures in cellular environments will ultimately lead to the development of new theranostic applications of nanomaterials.