This study modeled and optimized the operational parameters for biohydrogen production from waste sugarcane leaves and assessed hydrogen production on a semi-pilot scale. A Box-Behnken design with input variables of substrate concentration (8 to 24 g/L), inoculum concentration (10% to 50% v/v), and hydraulic retention time (HRT, 24 to 96 h) was used. A coefficient of determination (R2) of 0.90 and the predicted optimum operational set-points of 14.2 g/L substrate concentration, 32.7% inoculum concentration, and 62.8 h HRT were obtained. Experimental validation produced a biohydrogen yield of 12.8 mL H2/g fermentable sugar (FS). A semi-pilot scale process in a 13-L Infors reactor under optimized conditions gave a cumulative hydrogen volume and yield of 3740 mL and 321 mL H2 g-1 FS, respectively, with a peak hydrogen fraction of 37%. Microbial analysis from the process effluent conducted by Polymerase Chain Reaction cloning indicated the presence of hydrogen-producing bacteria belonging to Clostridium sp., Klebsiella sp., and Enterobacter sp. These findings highlight the feasibility of biohydrogen production from sugarcane waste and provide preliminary knowledge on process scale up.