A two-stage modelling and optimization of biohydrogen production is reported. A mixture design was used to determine the optimum proportions of bean husk (BH), corn stalk (CS), and organic fraction of solid municipal waste (OFSMW). The optimum operational set-points for substrate concentration, pH, temperature and hydraulic retention time (HRT) were further investigated using the box-behnken design. The quadratic polynomial model from the mixture design had a coefficient of determination (R-2) of 0.9427 and the optimized mixtures were in the ratio of OFSMW:BH:CS = 30:0:0 and OFSMW:BH:CS = 15:15:0 with yields of 56.47 ml H-2/g TVS and 41.16 ml H-2/g TVS respectively. Optimization on physicochemical process parameters on the improved substrate gave the setpoints of 40.45 g/l, 7.9, 30.29 degrees C, 86.28 h for substrate concentration, pH, temperature and HRT respectively having a predicted H-2 yield of 57.73 ml H-2/g TVS. Model validation gave 58.62 ml H-2/g TVS, thus an improvement of 3.8% on the optimized mixture. Biohydrogen production can be significantly enhanced by a suitable mixture of agro-municipal waste and operational optimal setpoints. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.