Anaerobic hydrogen fermentation converting carbohydrate-rich biomass to clean hydrogen energy is a renewable technology for hydrogen production. Applying immobilization technology on hydrogen fermentation could increase the cell concentration, hydraulic loading and organic loading. This study developed a novel technology to immobilize hydrogen producing bacteria in a thin film and attached on a carrier by polymer. The thin attached biofilm provides a stable hydrogen producing bacteria seeding source and makes the good hydrogen production performance. After 115 days, 9 generation transfer culture, the hydrogen yield could maintain a yield over 1.8 mol-H(2)/mol-hexose. Moreover, immobilized biofilm decreased the lag phase for proximately 65-70%. The bacteria wrapped in the immobilized biofilm could grow through the biofilm and then cover the surface of biofilm. The immobilized biofilm provides seed bacteria to bulk solution continuously. Therefore, immobilized biolfilm could be a promising immobilization technology and a good seeding source for biohydrogen fermentation. (C) 2008 Elsevier Ltd. All rights reserved.