The immobilization of proteins on inorganic supports is attracting increasing interest since the realization of active surfaces finds application in enzyme-assisted catalysis, environmental sciences, and medical fields. In the present study, cytochrome c (cyt c) is adsorbed on silica nanoparticles (SNPs) and amino-functionalized silica nanoparticles (SNPs-APTES), which are prepared for this purpose and having a diameter of about 50 nm. The peroxidase activity of the protein is investigated under different experimental conditions, to evaluate the impact of differently charged surfaces on the catalytic activity of the biomolecule. The peroxidase activity of cyt c increases upon adsorption on SNPs, and it shows a linear behavior with nanoparticles concentration; on the other hand, the contact with increasing amounts of SNPs-APTES does not affect the catalytic activity of the protein. The kinetic profile of the oxidation reaction is altered for cyt c-SNPs sample, suggesting that upon adsorption, changes in the catalytic process take place. Moreover, we observe that the enhancement of peroxidase activity of cyt c-SNPs is almost completely inhibited in high-ionic-strength buffer: this indicates that the protein establishes electrostatic interactions with SNP. The spectroscopic properties of the adsorbed protein on the two different matrices are investigated by using fluorescence and Raman spectroscopies to account for the enzymatic activity of the hybrid materials. The fluorescence spectra of cyt c-silica bio-composites reveal that the adsorption on silica modifies the microenvironments of the emitting amino acid residues of the protein. Indeed, their fluorescence gains intensity and appears blue-shifted compared to that of the native protein; these modifications are more evident when cyt c is adsorbed on SNPs. Raman spectra suggest that both oxidation and spin state of heme iron change when cyt c is adsorbed on SNPs but not on SNPs-APTES. The spectroscopic data of biocomposite materials are discussed in terms of structural changes to account for the increment of peroxidase activity upon adsorption on the negatively charged surface of SNPs.