Intelligent magnetic materials are capable of responding to environmental stimuli in an on/off manner. The photo- and thermal-responsive nanogel layer was surface-grafted onto several magnetic nanoparticles to prepare the magnetic imprinted nanoparticles (Fe3O4 MIPs). The Fe3O4 MIPs show dramatic temperature and light, dual-dependent rebinding and release characteristics for lysozyme. Above the volume phase transition temperature of the PNIPAAm, the imprinted protein (lysozyme) can be selectively separated from egg whites and the activity remained unchanged. By combining low temperature and UV irradiation, a more finely tuned release of the binding protein can be achieved. Time-dependent antibacterial activity was observed, and it was found that Fe3O4 MIPs-loaded lysozyme almost completely suppressed the growth of E. coli with the continuous lysozyme release. More importantly, the Fe3O4 MIPs could easily be separated by an external magnetic field and can be recycled. The photo- and thermal-responsive Fe3O4 MIPs pave a new way for the development of an advanced, sustainable material for use in bioseparation, drug delivery, and antibacterial materials.