This work aims to develop a repeatable enzyme entrapment method that preserves activity within an amicable environment while resisting activity reduction in the presence of environmental challenges. Advances in such methods have wide potential use in biosensor applications. In this work β-galactosidase (lactase) enzyme was entrapped within hydrogel matrices of acrylamide (ACR) crosslinked with N,N′-methylenebisacrylamide (BIS, non-degradable) or poly(ethylene glycol) diacrylate (PEGDA, degradable) to create “biogels.” Diffusivity studies of control, enzyme free, hydrogel constructs showed near-Fickian swelling behavior in PBS regardless of crosslinker type or density. As expected, the swelling rate, Ks, decreased when increasing the crosslink density from 78.6 to 14.7 min−1 over a range of 1–20 mol% PEGDA indicating that diffusivity into the matrix is dependent on crosslink density. Fabricated biogels were evaluated for maintained enzyme activity in the 7 and 8 pH range. PEGDA crosslinked gels consistently showed improved enzymatic activity retention as compared to BIS crosslinked gels. As PEGDA crosslink density increased from 5 to 10 mol%, enzymatic activity retention post-initial entrapment increased. Higher PEGDA crosslink densities between 15% and 40% decreased enzymatic activity due to assumed steric hindrance of the entrapped enzyme and also decreased substrate and product diffusion. Increased enzymatic stability was observed in 40 mol% PEGDA crosslinked gels. The biogels were pH challenged to 8.0 and stability, measured as retention of activity, was observed to be 91%. Free, non-entrapped, solution based enzyme conversion only retained 23% activity under the same pH challenge conditions. No significant loss of active enzyme was determined to elute out of the biogels during storage in PBS or during biogel wash and recycling. This entrapment method illustrates the potential to sterically hinder and diffusively impede enzymes from performing their function. Degradation of the network crosslinks can then potentially enable the reactivation of the enzyme at a site and time dictated by the user. Biotechnol. Bioeng. 2013;110: 2994–3002. © 2013 Wiley Periodicals, Inc.