A novel metal composite material based on zirconium dioxide decorated gold nanoparticles (ZrO2@AuNPs), copper (I)oxide at manganese (IV) oxide (Cu2O@MnO2) and immobilized choline oxidase (ChOx) onto a glassy carbon electrode(GCE) (ChOx/Cu2O@MnO2-ZrO2@AuNPs/GCE) has been developed for enhancing the electro-catalytic property,sensitivity and stability of the amperometric choline biosensor. The ChOx/Cu2O@MnO2-ZrO2@AuNPs/GCE displayedan excellent electrocatalytic response to the oxidation of the byproduct H(2)O(2)from the choline catalyzed reaction,which exhibited a charge transfer rate constant (K-s) of 0.97 s(-1), a diffusion coefficient value (D) of 4.50x10(-6) cm(2)s(-1), an electroactive surface area (A(e)) of 0.97 cm(2)and a surface concentration (gamma) of 0.54x10(-8) mol cm(-2).The modified electrode also provided a wide linear range of choline concentration from 0.5 to 1,000.0 mu M with goodsensitivity (97.4 mu A cm(-2) mM(-1)) and low detection limit (0.3 mu M). The apparent Michaelis-Menten constant wasfound to be 0.08 mM withI(max)of 0.67 mu A. This choline biosensor presented high repeatability (%RSD=2.9, n=5),excellent reproducibility (%RSD=2.9, n=5), long time of use (n=28 with %I>50.0 %) and good selectivity withoutinterfering effects from possible electroactive species such as ascorbic acid, aspirin, amoxicillin, caffeine, dopamine,glucose, sucrose and uric acid. This optimal method was successfully applied for choline measurement in preparedhuman blood samples which demonstrated accurate and excellent reliability in the recovery range from 96.7 to 102.0%.