Inspired by adhesive proteins secreted by mussels, a general biomolecule immobilization approach has been proposed to the synthesis of multifunctional core-shell heme protein-Au-polydopamine-Fe3O4 magnetic bionanoparticles (heme protein-Au-PDA-Fe3O4 MBNPs) using one-pot chemical polymerization. Scanning electron microscope, energy dispersive X-ray spectrometer, UV-vis spectroscopy, and electrochemical methods were used to characterize the heme protein-Au-PDA-Fe3O4 MBNPs. Results demonstrated that the resultant MBNPs not only had the magnetism of Fe3O4 nanoparticles which made them easily manipulated by an external magnetic field, but also had the excellent biocompatibility and good conductivity of PDA and Au nanoparticles which could maintain the native structure of the heme proteins, and facilitated the direct electrochemistry of heme proteins including hemoglobin (Hb), myoglobin (Mb) and horseradish peroxidase (HRP) in the biofilm. Hence, the protein-Au-PDA-Fe3O4 biofilm electrode displayed excellent catalytic performance for H2O2. This facile, versatile, and inexpensive proteins immobilization platform is promising for the preparation of many other MBNPs with interesting properties and application potentials in many fields, such as biosensing, biocatalysis, biofuel cells, and bioaffinity separation. (C) 2013 Elsevier B.V. All rights reserved.