In this work, a novel bio-carrier was used to immobilize photo-fermentative bacteria for hydrogen production. The results showed that the bacterial immobilization and hydrogen production were strongly affected by particle size, amount of bio-carrier and light intensity. Controlling the proper size of bio-carrier not only prevented light shading effect from each other, but also made solid carriers better fluidized during operation. The scanning electronic microscopy revealed that the biofilm formed by photo-fermentative bacteria on the surface of bio-carrier enhanced hydrogen production. Because of bio-carrier fluidization, each immobilized bacterium could receive light energy and produce hydrogen. With the optimal particle size (2 x 2 mm), amount (3% weight to volume ratio) and light intensity (6000 lux), the maximum hydrogen yield of 3.24 mol H-2/mol acetate and production rate of 36.06 ml/Vh were obtained in continuous operation stage. Bio-carrier was an effective solid carrier to immobilize photo-fermentative bacteria for improving hydrogen production. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.