As a perennial grass, Humulus scandens is rich in cellulose which can be fermented for bioenergy producing. Hence, the hydrogen production potential of the Humulus scandens from dark fermentation by Enterobacter aerogenes was investigated in this paper. Cellulase amount, inoculation amount, and initial pH value were evaluated. The interrelationship between these factors were studied by Response Surface Box-Behnken Experiments. Results showed that there was a significant correlation between the three factors. Through the correction of the regression equation, the optimized technological conditions were obtained. The amount of cellulase was 0.203 g g(-1) TS, the inoculation amount was 42.6%, the initial pH value was 6.59, the pre-estimated maximum cumulative value of hydrogen production was 65.12 mL g(-1) TS, and it was similar to the test mean value which was 64.08 mL g(-1) TS. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.