Magnetic single-enzyme nanoparticles (MSENs) encapsulated within a composite Fe3O4/poly(pyrrole-N-propylsulfonic acid) network were successfully fabricated via the surface modification and in situ aqueous polymerization of separate enzyme molecules. The resulting MSENs with magnetism and conductivity exhibited high activity and stability. An amperometric glucose biosensor has been developed based on the modification of MSENs onto magnetic glassy carbon electrode (MGCE) through magnetism immobilization. The performance of the developed glucose biosensor was evaluated and the results indicated that a sensitive glucose biosensor could be fabricated. The obtained glucose biosensor presents high-selectivity monitoring of glucose, satisfactory stability, fast response time (4 s), wide linear range (0.0005-3.5 mM) and low detection limit (0.2 mu M). The analytical application of the glucose biosensor confirms the feasibility of glucose detection in serum sample. (C) 2013 Elsevier Ltd. All rights reserved.