An effective, stable enzymatic glucose biosensor was fabricated on a glassy carbon electrode (GCE) surface using simple multicomposite materials (MCM): a solution of prepared poly(diallyldimethylammonium chloride)‐capped gold nanoparticles‐nickel ferrite particles‐carbon nanotubes‐chitosan (PDDA‐AuNPs‐NiFe2O4‐CNTs‐CHIT), electropolymerization of poly(o‐phenylenediamine) (PoPD) and immobilization of glucose oxidase (GOx). Biocompatibility and synergy of the MCM enhanced the immobilization and the reaction of GOx and as well as the electron transfer from an oxidation reaction of hydrogen peroxide in the system. The NiFe2O4 was synthesized by co‐precipitation and calcined at 700 °C. Characterization was carried out by field emission scanning electron microscopy (FE‐SEM), energy‐dispersive X‐ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR) and X‐ray diffraction (XRD) which presented both tetrahedral and octahedral metal stretching with a cubic NiFe2O4 crystal phase. The GOx/PoPD/MCM/GCE yielded a 0.77 s−1 charge transfer rate constant (Ks), a 2.28×10−6 cm2 s−1 diffusion coefficient value (D), a 0.21 mm2 electroactive surface area (Ae) and a 1.93×10−8 mol cm−2 surface concentration (γ ) as determined by cyclic voltammetry. The modified electrode showed a durable operation time (n=97, more than 50 % I), repeatability (%RSD=0.38, n=10), reproducibility (%RSD=1.60, n=10), high sensitivity (853.07 μA mM−1 cm−2), selectivity without effects of electroactive species (aspirin, uric acid, caffeine, cholesterol, ascorbic acid and dopamine) and two linear ranges from 0.5 to 10 μM (R2=0.998) and 10 to 15,000 μM (R2=0.991) with a low detection limit (0.35 μM, S/N=3). Its Michaelis‐Menten constant (Km) was calculated as 93.51 μM with 46.30 μA maximum current (Imax). This proposed simple method was successfully applied for glucose determination in human blood samples.