Enzyme immobilization has been the focus of extensive research over decades, yet empiricism still often prevails over rational design. To overcome this pattern and develop high performance, cost-effective systems, a trend towards a more comprehensive characterization of the heterogeneous bioconversion systems is emerging. This work encompasses such scope. The model system is the immobilization of invertase on glass substrate for the production of invert sugar syrup through sucrose hydrolysis. Before invertase immobilization, the glass substrates were cleaned and hydroxylated; next, substrate functionalization was performed by incubation with (3-Aminopropyl)triethoxysilane and glutaraldehyde. Detailed characterization was performed by monitoring every step of the immobilization protocol by contact angle measurement, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy. Enzyme loading and affinity towards the functionalized substrate was evaluated with a quartz crystal microbalance. The successful deposition of the different coating reagents and of the enzyme was confirmed by the shifts in surface wettability and roughness. XPS highlighted the over-simplistic nature of common models for chemical interaction between layers. Surface saturation was observed for the protein concentration of 0.5 mg/mL alongside with surface coverage of 910 ngenzyme/cm2. Finally, the suitability of the enzyme immobilization protocol was validated through operation in a packed bed reactor.