Isolation of specific cells from biological samples is an important aspect of various biological research and diagnostic applications. Magnetic separation using magnetic particles (MPs) allows for easy and specific isolation of the target cells. However, depending on the target cell antigen, biological ligands, such as antibodies, must be modified or altered on MPs. Additionally, further biological evaluation of isolated cells requires the removal of MPs from cells by the enzymatic degradation of the biological ligands. In this study, we designed a magnetic cell separation system in which temperature-responsive protein nanoparticles mediated the interaction between target cells and MPs, achieving the easy changeability of biological ligands, removal of MPs by cooling, and effective cell isolation. The protein nanoparticles were thermally responsively formed from fusion proteins constituted of elastin-like polypeptide (ELP), poly(aspartic acid) [poly(d)], and proteins-of-interest such as NanoLuc luciferase (Nluc) fused with replication initiation protein (Rep) (ELP-poly(d)-Nluc-Rep) or biotin acceptor peptide (BAP) (ELP-poly(d)-Nluc-BAP). Rep exhibited enzymatic conjugation activity with an optional DNA aptamer to protein nanoparticles. The transmembrane glycoprotein mucin 1 (MUC1)-binding DNA aptamer was conjugated to Rep as a model aptamer. Bioluminescence signals emitted from the Nluc domains were used to analyze the binding abilities. BAP contributed to binding to streptavidin-modified MPs via a biotin-streptavidin interaction. The MUC1-conjugated protein nanoparticles bound to MUC1-positive human breast cancer MCF-7 cells via MUC1 aptamers and streptavidin-conjugated MPs via BAP, leading to magnetic cell separation. The ratio of isolated MCF-7 cells via magnetic separation was 71.3% for the MCF-7 suspension at 1000 cells/1 mL. The MPs bound on recovered MCF-7 cells were removed by cooling at 4 °C to induce the dissociation of protein nanoparticles. Magnetic cell separation systems that use protein nanoparticles are a promising technology for biological research and diagnostic applications.