The ZnO nanoparticles (ZnONPs) were synthesized with gelatin as stabilizer via the sol-gel method and were characterized by transmission electron microscope (TEM), scanning electron microscopy (SEM) and X-ray diffraction (XRD). An electrochemical sensor based on ZnO nanoparticles-multi wall carbon nanotubes-poly methyl metacrylat (ZnONPs-MWCNT-PMMA) composite film was developed by incorporating Ni2+ into the ZnONPs-MWCNT-PMMA film modified carbon paste electrode (Ni2+/ZnONPs-MWCNT-PMMA/CPE). The electrochemical activity of Ni2+/ZnONPs-MWCNT-PMMA/CPE was illustrated in 0.10 M NaOH using cyclic voltammetry. The Ni2+/ZnONPs-MWCNT-PMMA/CPE exhibits the characteristic of improved reversibility and enhanced current responses of the Ni(III)/Ni(II) couple. Ni2+/ZnONPs-MWCNT-PMMA/CPE also show good electrocatalytic activity toward the oxidation of carbohydrates (glucose, fructose and sorbitol). The Ni2+/ZnONPs-MWCNT-PMMA/CPE gives a good linear range with a detection limit of 8, 6, and 9 μM towards the determination of glucose, fructose and sorbitol, respectively by amperometry. Furthermore, the modified sensor was successfully applied to the sensitive determination of carbohydrates in real samples.