A highly sensitive and stable glucose sensor based on the synergic action of multi wall carbon nanotube (MWCNTs) and ZnO nanoparticles was developed. Glucose oxidase (GOx) was firmly immobilized on the ZnO nanoparticles surface due to their large difference in the isoelectric point (IEP), while the ZnO nanoparticles were deposited on the negatively charged MWCNTs layer. A cationic polydiallyldimethylammonium chloride (PDDA) layer was coated on the GOx layer. The unique sandwich-like layer structure (PDDA/GOx/ZnO/MWCNTs) formed by self-assembling provides a favorable microenvironment to keep the bioactivity of GOx and to prevent enzyme molecule leakage. Amperometric detection of glucose was carried out at 100 mV (vs. Ag/AgCl) in 0.05 mol/L phosphate buffer solution (pH 6.8); a wide linear response range of 0.1 to 16 mmol/L was measured by a sensor with 0.5 units GOx; a sensitivity of 50.2 mA#8226;cm-2#8226;mol-1 and a detection limit of 250 nmol/L (3σ) were measured by a sensor with 2.0 units GOx. Results obtained from the glucose sensor directly in human blood serum were compared with ones obtained by spectrometry by a local hospital. On testing 100 samples of serum, the correlation coefficient reaches 0.997.