Multiwalled carbon nanotubes (MWNTs) grafted chitosan (CS) nanowire (NW) was prepared by phase separation method. Glucose oxidase (GOx) was sequentially immobilized into MWNT-CS-NW to obtain MWNT-CS-NW/GOx biosensor. Field emission scanning electron microscopy (FESEM) images of MWNT-CS-NW/GOx reveals the existence of MWNT and CS. Cyclic voltammetry and amperometry were used to evaluate the electrochemical determination of glucose. The MWNT-CS-NW/GOx biosensor shows an excellent performance for glucose at +0.34V with a high sensitivity (5.03μA/mM) and lower response time (3s) in a wide concentration range of 1–10mM (correlation coefficient of 0.9988). In addition, MWNT-CS-NW/GOx biosensor possesses better reproducibility, storage stability and there is negligible interference from other electroactive components.