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Biomolecule immobilization on bulk silicon dioxide (SiO2) is an important aspect in the field of Si-based interfaces for biosensing. The approach used for surface preparation should guarantee not only the stable anchoring of biomolecules but also their structural integrity and biological functioning. In this paper, we review our findings on the SiO2 functionalization process to immobilize a variety of biomolecules, including glucose oxidase, horseradish peroxide, metallothionein, and DNA molecules. Morphological and chemical characterization of SiO2 surfaces after biomolecule immobilization using techniques already employed in the microelectronic industry are presented and discussed. Optical and spectrophotometric analysis revealed the preservation of biomolecules’ activity once they are anchored on the biointerface.