Background: Hydrogen is regarded as an attractive future energy carrier for its high energy content and zero CO 2 emission. Currently, the majority of hydrogen is generated from fossil fuels. However, from an environmental perspective, sustainable hydrogen production from low-cost lignocellulosic biomass should be considered. Thermophilic hydrogen production is attractive, since it can potentially convert a variety of biomass-based substrates into hydrogen at high yields. Results: Sugarcane bagasse (SCB) was used as the substrate for hydrogen production by Thermoanaerobacterium aotearoense SCUT27/Δldh. The key parameters of acid hydrolysis were studied through the response surface methodology. The hydrogen production was maximized under the conditions of 2.3% of H 2 SO 4 for 114.2 min at 115°C. Using these conditions, a best hydrogen yield of 1.86 mol H 2 /mol total sugar and a hydrogen production rate (HPR) of 0.52 L/L · h were obtained from 2 L SCB hydrolysates in a 5-L fermentor, showing a superior performance to the results reported in the literature. Additionally, no obvious carbon catabolite repression (CCR) was observed during the fermentation using the multi-sugars as substrates.