BACKGROUND: The main problem when immobilizing enzyme inside hydrogel is the activity loss of the trapped enzymes due to the limitation on diffusion. In this study, macroporous poly(ethylene glycol) (PEG) hydrogel membrane was fabricated using photolithography to enhance the activity of immobilized enzyme. RESULT: Photopolymerization through the designed photomask produced crosslinked hydrogel with tunnel-like macropores which were completely opened from top to bottom of the hydrogel membranes. Arrays of pores with 50 mu m and 100 mu m diameters were successfully created in the hydrogel membranes, and the pore density could be controlled by changing the photomask design. An activity study demonstrated that there was an enhancement of more than 50% in glucose oxidase activity in the macroporous membrane in comparison to the nonporous membrane. The activity of immobilized enzyme could be further enhanced by increasing the pore density of the hydrogel membranes or by using a higher molecular weight PEG. CONCLUSION: Increased enzymatic activity was observed in macroporous hydrogel membranes compared with nonporous hydrogel membranes due to the higher surface to volume ratio provided by the macroporous structure. Compared with other methods of obtaining porous hydrogel, the proposed method is much simpler and can minimize the deactivation of enzyme. (C) 2007 Society of Chemical Industry.