Sphere-capped ferrocene nanospheres with Schiff base spacers have been prepared using a template, and used as carriers to immobilize glucose oxidase (GOx). GOx immobilized on spheres with one C-spacer (APS-Fc) exhibited high binding affinity to the substrate, which was attributed to appropriate position for the GOx conformation. When glucose oxidase was immobilized with spacers of different lengths, it was found that storage stability decreased with increasing the length of the spacer. It has been found that nanospheres, including capped ferrocene, exhibit good performance as the immobilized supporters of GOx. (APS-EtFc-GOx) retain more than 10% of the initial activity after forty-two successive cycles, which is a remarkable result.