N,N′‐bis(2‐hexyl)‐2,6‐(3,4 ethylenedioxythiophene)‐1,4,5,8‐naphthalenimide (ENDI) monomer was electropolymerized on pencil graphite electrode (PGE) and used as a biosensor for glucose immobilization for the first time. After electropolymerization of ENDI monomer on PGE (AuNPs/PENDI/PGE), the intensity of the current signal was improved by using gold nanoparticles (AuNPs). The immobilization of the glucose oxidase (GOx) enzyme was carried out by using the cross‐linking reagent glutaraldehyde (GA) due to the strong π‐π* interaction of the poly‐ N,N′‐bis(2‐hexyl)‐2,6‐(3,4 ethylenedioxythiophene)‐1,4,5,8‐naphthalenimide (PENDI) polymer. GOx/AuNPs/PENDI/PGE designed biosensor, was characterized by a wide linear glucose determination range between 0.0009–0.33 mM with a limit of detection of 0.0407 mM and a sensitivity of 0.172 μA/mMcm2. Herein we have shown that GOx/AuNPs/PENDI/PGE biosensor effectively provides the enzyme immobilization and very effective for glucose determination in commercial samples.