The application of the composites of multiwall carbon nanotubes (MWNTs) and core-shell organosilica@chitosan crosslinked nanospheres as an immobilization matrix for the construction of an amperometric hydrogen peroxide (H2O2) biosensor was described. MWNTs and positively charged organosilica@chitosan nanospheres were dispersed in acetic acid solution (0.6wt%) to achieve organosilica@chitosan/MWNTs composites, which were cast onto a glass carbon electrode (GCE) surface directly. And then, horseradish peroxidase (HRP), as a model enzyme, was immobilized onto it through electrostatic interaction between oppositely charged organosilica@chitosan nanospheres and HRP. The direct electron transfer of HRP was achieved at HRP/organosilica@chitosan/MWNTs/GCE, which exhibited excellent electrocatalytic activity for the reduction of H2O2. The catalysis currents increased linearly to H2O2 concentration in a wide range of 7.0 x 10(-7) to 2.8 x 10(-3) M, with a sensitivity of 49.8 mu A mM(-1) cm(-2) and with a detection limit of 2.5 x 10(-7) M at 3 sigma. A Michaelies-Menten constant K-M(app) value was estimated to be 0.32 mM, indicating a high-catalytic activity of HRP. Moreover, the proposed biosensor displayed a rapid response to H2O2 and possessed good stability and reproducibility. When used to detect H2O2 concentration in disinfector samples and sterilized milks, respectively, it showed satisfactory results. (C) 2008 Elsevier Ltd. All rights reserved.