The present study developed a two-step continuous hydrogen production process using immobilized mixed culture with corn stalk as adsorption carrier. The carrier inoculum and hydraulic retention time (HRT) were optimized in batch and semi-continuous tests, respectively. The results showed that the immobilized bacteria could use starch directly, and the highest hydrogen yield of 1.81 mol H-2/mol glucose was obtained with a carrier inoculum of 1% in the batch test. The semi-continuous test exhibited a continuous and stable hydrogen production for 20 days at HRT = 48 h, with an average hydrogen production rate of 223.56 mL/L/d. Under these conditions, a two-step continuous test was successfully developed, and the hydrogen production in both two reactors increased linearly for 18 days without any start-up period. The total hydrogen production rate could reach 429.72 mL/L/d. The results indicated the potential of using corn stalk as adsorption carrier for continuous hydrogen production. (C) 2018 Elsevier Ltd. All rights reserved.