NiCu alloys have been suggested as potential candidates for catalysts in glucose oxidation. In this study, NiCu alloys with different compositions were prepared on a glassy carbon substrate by changing the electrodeposition potential to examine the effect of Ni/Cu ratios in alloys on catalytic activity toward glucose oxidation. Cyclic voltammetry and chronoamperometry showed that NiCu alloys had higher catalytic activity than pure Ni and Cu catalysts. Especially, Ni59Cu41 had superior catalytic activity, which was about twice that of Ni at a given oxidation potential. X-ray analyses showed that the oxidation state of Ni in NiCu alloys was increased with the content of Cu by lattice expansion. Ni components in alloys with higher oxidation state were more effective in the oxidation of glucose.