The Bed Pressure Fluctuation Analysis (PFA) and dual electrical resistivity probe methods were used to investigate the hydrodynamic properties in a bioreactor. It was found that the dominant frequency and side frequency signals were not obvious from Hydraulic Retention Time (HRT) 4 to 0.5 h on the power spectrum diagram when using condensed molasses fermentation soluble (CMS) as the substrate in a biohydrogen production reactor. The start-up testing and online monitoring of the bed pressure fluctuation in a pilot system with HRT 12 h was also investigated. The pressure fluctuation analysis method established a hydrodynamic behavior diagram that was effective in determining flow regime of biogas in large-scale bioreactors. Finally, the study found that a mathematical model built using MATLAB to calculate and simulate the dynamic behavior of bubbles in a biological hydrogen production reactor satisfactorily. The bubble rise velocity and bubble size increased from 0.7 to 1.5 cm/s and 0.2 to 1.9 x 10(-6) cm respectively when the biogas production rate was increased from 30 to 70 mL/min in the biohydrogen production reactor. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.