Polyvinyl alcohol (PVA) is a water-soluble, biocompatible and biodegradable synthetic polymer whose application in the immobilization of biological agents for use in biocatalysis has shown promising results. This study aimed to investigate and optimize the immobilization of naringinase from Penicillium decumbens in PVA networks, targeting for the hydrolysis of naringin. Variables such as the most suitable cross-linker, catalyst, inorganic salt, co-solvents and solidification process were identified as key issues for PVA-based methods to form lens-shaped particles, while retaining high enzyme activity and stability. Major improvements were established for better and more reproducible immobilization conditions, namely, by designing a new immobilization apparatus to produce uniform lens-shaped particles. The common problems of PVA-based entrapment were significantly mitigated, through the use of selected cross-linker, glutaraldehyde (GA), and co-solvent, dimethyl sulfoxide (DMSO), which decreased the toxicity of the immobilization process and allowed the control of membrane porosity, respectively. The relevance of DMSO and GA and their interaction and effect on the swelling ratio, encapsulation efficiency and residual activity of PVA biocatalysts were established. The immobilization of naringinase in PVA under a DMSO concentration of 60%, cross-linked with 1% GA, and particle lens size of 3.5–4.0 mm, width of 100–300 µm and average particle volume of 12.5 ± 0.92 µL, allowed an encapsulation efficiency of 98.6% and an average residual activity of 87% ± 3.6%. The kinetic characterization of the immobilized naringinase showed no changes in pH profile, whereas hydrolytic activity increased up to 60 °C. Immobilization in PVA/DMSO/GA lens-shaped particles enhanced the storage stability of naringinase. Moreover, these naringinase bio-immobilizates retained a conversion rate higher than 78% after 23 runs. Copyright © 2012 John Wiley & Sons, Ltd.