An unsaturated flow bioreactor with Rhodopseudomonas palustris CQK 01 was developed for photo-hydrogen production. The bioreactor developed in this work consisted of a reaction bed packed with spherical glass beads and internal optical fiber that provided uniform light distribution and achieved high light transmission efficiency. Unsaturated conditions were obtained by filling both argon and nutrient solution into the bioreactor. Experimental results demonstrated the feasibility of the biofilm formation in an unsaturated environment. With the formed biofilm, parametric studies on the photo-hydrogen production performance under different liquid flow rates and initial substrate concentrations were conducted. It was found that the performance of the bioreactor came to the optimal state at the liquid flow rate of 500 mL/h. Moreover, such a design enabled the bioreactor to be operated at relatively high glucose concentration without the substrate inhibition, yielding a good biohydrogen production performance. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.