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Polyaniline–mutilwalled carbon nanotube (PANi–MWCNT) nanocomposites were electropolymerized in the presence of sodium dodecyl sulfate (SDS) onto interdigitated platinum-film planar microelectrodes (IDμE). The MWCNTs were first dispersed in SDS solution then mixed with aniline and H2SO4. This mixture was used to electro-synthesize PANi–MWCNT films with potentiostatic method at E = + 0.90 V (versus SCE). The PANi–MWCNT films were characterized by cyclic voltammetry (CV) and scanning electron microscopy (SEM). The results show that the PANi–MWCNT films have a high electroactivity, and a porous and branched structure that can increase the specific surface area for biosensing application. In this work the PANi–MWCNT films were applied for covalent immobilization of glucose oxidase (GOx) via glutaraldehyde agent. The GOx/PANi–MWCNT/IDμE was studied using cyclic voltammetric and chronoamperometric techniques. The effect of several interferences, such as ascorbic acid (AA), uric acid (UA), and acetaminophen (AAP) on the glucosensing at +0.6 V (versus SCE) is not significant. The time required to reach 95% of the maximum steady-state current was less than 5 s. A linear range of the calibration curve for the glucose concentration lies between 1 and 12 mM which is a suitable level in the human body.