This study assessed the effect of hydraulic retention time (HRT) on biohydrogen production by dark fermentation using an anaerobic fluidized bed reactor fed with cheese whey synthetic wastewater. The reactor of 1.2 L was operated with an approximate carbohydrate concentration of 2800 mg L -1 , and HRTs of 6, 4, 2, 1 and 0.5 h. Acetic and butyric acids were the main metabolites produced in the reactor. The hydrogen production increased from 0.039 to 1.43 L-H 2 h -1 L -1 -reactor as the HRT decreased from 6 to 0.5 h. The best hydrogen yield (HY) (2.73 mol-H 2 mol-carbohydrate -1 ) and carbohydrate consumption (81.02%) were achieved at a HRT of 6 h (12.34 kg-COD m -3 d -1 ) followed by the HY of 2.43 mol-H 2 mol-carbohydrate -1 at a HRT of 0.5h (163.02 kg-COD m -3 .d - 1 ). An increase in the influent pH in the operational phase 05 (HRT of 0.5 h) favored hydrogen production despite the reduced carbohydrate conversion compared to phase 01 (HRT of 6 h). In both conditions, the reactor presented the highest amount of acetic acid, indicating that the acetate route favored the hydrogen yield production. The HRT reduction led to an increase in microbial diversity, as evidenced by the Shannon-Wiener coefficient of 2.586, which corresponds to the operational phase with a HRT of 2 h.