The current state of palm oil mill wastewater treatment focuses solely on open ponding or closed lagoon systems for biogas production. However, efforts to convert this wastewater into biohydrogen are limited. Therefore, this research investigates the feasibility of converting palm oil mill effluent (POME) for biohydrogen production via dark fermentation. Temperature and hydraulic retention time (HRT) effects on biohydrogen production and COD removal efficiency in an up-flow anaerobic sludge fixed-film (UASFF) bioreactor were investigated. The experiment was carried out and analysed using a central composite design (CCD) and the Response Surface Methodology (RSM). The hydrogen (H 2 ) yield, H 2 production rate (HPR), and COD removal efficiency were investigated as responses. HPR increased significantly by 28.8 folds as temperature increased from 37 °C to 53.5 °C (transition from mesophilic to thermophilic) at HRT of 3 h. Meanwhile, the COD removal efficiency significantly increased from 24.76% to 33.33% between 4 to 9 h of HRT. Maximum H 2 yield of 0.95 L H 2 g −1 COD removed , HPR of 10.39 L H 2 d −1 , and 35.9% COD removal were reported at the optimum HRT and temperature of 7 h and 57 °C, respectively. This study indicates that under the thermophilic condition and short HRT, POME could be treated while producing biohydrogen using the UASFF bioreactor. • The temperature has a significant impact on hydrogen yield and hydrogen production rate. • Low HRT significantly impacted the COD removal efficiency of POME. • Optimum temperature and HRT were 57 °C and 7 h, respectively. • Acetobacter spp. is found to be dominant under a thermophilic condition in the H 2 -UASFF unit.