A novel needle-type biosensor based on carbon nanotubes is reported. Thebiosensor was prepared by packing a mixture of multi-wall carbon nanotubes (MWCNTs),graphite powder and glucose oxidase (Gox) freeze-dried powder into a glass capillary of 0.5mm inner diameter. The resulting amperometric biosensor was characterizedelectrochemically using amperometry in the presence of hydrogen peroxide and in thepresence of glucose. The glucose biosensor sensitivity was influenced by the glucoseoxidase concentration within the MWCNTs mixture. The optimized glucose needle-typebiosensor displayed better sensitivity and stability, and a detected range of up to 20 mM.Based on its favorable stability, the needle biosensor was first time used in real-timemonitoring system as a kind of online glucose detector. The decay of current response isless than 10% after 24-hour continuous observation.