In this work, we have compared the entrapment of free or previously immobilized glucose oxidase using a sol-gel technique. The preimmobilization was carried out on Sepabeads (a porous support) derivatized with glutaraldehyde as the functional group. The prior immobilization of the enzyme permitted to maintain the enzyme activity intact after the formation of the sol-gel. In fact, only 10% of the enzyme activity was lost whereas the soluble enzyme lost 60% of its initial activity. Additionally, enzyme leakage from the sol-gel matrix was avoided, which was relatively high when entrapping the soluble enzyme (39% of the enzyme activity was released after 16 h of incubation in a buffered solution). Moreover, the immobilized enzyme, inside the porous support, cannot be in contact with the sol-gel, and, therefore, it maintained the stability achieved by means of the multipoint covalent attachment on the Sepabeads support.