Abstract: In order to find appropriate heat pretreatment methods for enriching hydrogen-producing bacteria from activated sludge, the shaking flask fermentation test was adopted to investigate the characteristics of fermentative hydrogen production from glucose in the wastewater treatment plant activated sludge under different heat pretreatment at 65 ℃. The results showed that after 64 hours of culture at 35 ℃ with an initial glucose concentration 10 g/L, seed sludge volume in terms of mixed liquor volatile suspend solid (MLVSS) of 1.6 g/L and pH of 7.0, and then respectively after 15 min and 30 min of heat pretreatment at 65 ℃, the seed sludge fermentation system illustrated good hydrogen production performance. The hydrogen-producing ratio from glucose and specific hydrogen yield in terms of MLVSS after 30 min of heat pretreatment reached 1.09 mol/mol and 8.4 mmol/g MLVSS, respectively, while that after 15 min of heat pretreatment reached 1.06 mol/mol and 8.2 mmol/g MLVSS, respectively. Additionally, different microorganisms were screened from the seed sludge under different heat pretreatment time, and the predominant acidogenic fermentative bacteria with different functions were constructed. Butyric acid type fermentation and mixed acid fermentation were established for the seed sludge fermentation system after 15-75 min and 90-120 min of heat pretreatment, respectively.