Abstract A combined system based on multiple steam explosion pretreatments (direct pretreatment and presoaked in 1% KOH aqueous solution followed by pretreatment under different conditions) and mild alkaline post-treatment has been developed to obtain digestible substrates from bamboo stems for bioethanol production. After the pretreatments, the enzymatic digestibility of cellulose increased to 17.1–32.2%, as compared to that of the untreated bamboo stems (5.8%). Direct pretreatment followed by alkaline treatment increased the enzymatic digestibility of cellulose to a maximum value of 73.8%. Alkaline treatment removed most of lignin and hemicelluloses, and incurred a higher crystalline index of the cellulose-rich residue obtained after a synergistic treatment as compared to the only steam-exploded substrates. The combination of direct pretreatment and alkaline treatment is an environmentally friendly and economical feasible method for the production of glucose and high-purity lignin, which will be further converted into high value-added products based on biorefinery.