Enzymatic bioreactors with both high flow characteristics and mechanical stability based on macroporous poly(2-vinyl-4,4-dimethylazlactone-co-acrylamide-co-ethylene dimethacrylate) monoliths have been prepared. Covalent immobilization of trypsin on these support is achieved in a single reaction step using the azlactone functional groups. Optimization of hydrophilic/hydrophobic properties of the monolith affords a support that does not shrink in water and leads to immobilized enzyme that shows high activity in the hydrolysis of both low and high molecular weight substrates such as L-benzoyl arginine ethyl ester and casein. The catalytic activity of the monolithic reactor is maintained even at a flow velocity of 180 cm/min, which substantially exceeds those reported in the literature for packed bed reactors.