Magnetic separation provides a relatively quick and easy-to-use method for cell isolation and protein purification. We have developed a rapid and efficient procedure to isolate yeast cells displaying a target polypeptide, namely, the Staphylococcus aureus ZZ domain, which serves as s model for protein interactions and can bind immunoglobulin G (IgG). We optimized selection of ZZ-displaying yeast cells using thermoresponsive magnetic nanoparticles. A model library was prepared by mixing various proportions of target yeast displaying the ZZ domain with control cells. Target cells in the model library that bound to the ZZ-specific binding partner, biotinylated IgG, were selected with biotinylated thermoresponsive magnetic nanoparticles using the biotin-avidin sandwich system. We determined ZZ expression levels and optimized the concentrations of both magnetic nanoparticles and avidin for efficient selection of target cells. After optimization, we successfully enriched the target cell population 4700-fold in a single round of selection. Moreover, only two rounds of selection were required to enrich the target cell population from 0.001% to nearly 100%. Our results suggest that magnetic separation will be useful for efficient exploration of novel protein-protein interactions and rapid isolation of biomolecules with novel functions.