ZnO:Co nanoclusters were synthesized by nanocluster-beam deposition with averaged particle size of 5 nm and porous structure, which were for the first time adopted to construct a novel amperometric glucose biosensor. Glucose oxidase was immobilized into the ZnO:Co nanocluster-assembled thin film through Nafion-assisted cross-linking technique. Due to the high specific active sites and high electrocatalytic activity of the ZnO:Co nanoclusters, the constructed glucose biosensor showed a high sensitivity of 13.3 microA/mA cm2. The low detection limit was estimated to be 20 microM (S/N=3) and the apparent Michaelis-Menten constant was found to be 21 mM, indicating the high affinity of the enzyme on ZnO:Co nanoclusters to glucose. The results show that the ZnO:Co nanocluster-assembled thin films with nanoporous structure and nanocrystallites have potential applications as platforms to immobilize enzyme in biosensors.