Background: To increase the production of bioethanol, a two-step process based on hydrothermal and dilute alkaline treatment was applied to reduce the natural resistance of biomass. However, the process required a large amount of water and a long operation time due to the solid/liquid separation before the alkaline treatment, which led to decrease the pure economic profit for production of bioethanol. Therefore, four one-step processes based on order of hydrothermal and alkaline treatment have been developed to enhance concentration of glucose of wheat straw by enzymatic saccharification. The aim of the present study was to systematically evaluated effect for different one-step processes by analyzing the physicochemical properties (composition, structural change, crystallinity, surface morphology, and BET surface area) and enzymatic saccharification of the treated substrates. Results: In this study, hemicelluloses and lignins were removed from wheat straw and the morphologic structures were destroyed to various extents during the four one-step processes, which were favorable for cellulase absorption on cellulose. A positive correlation was also observed between the crystallinity and enzymatic saccharification rate of the substrate under the conditions given. The surface area of the substrate was positively related to the concentration of glucose in this study. As compared to the control (3.0 g/L) and treated substrates (11.2-14.6 g/L) obtained by the other three one-step processes, the substrate treated by one-step process based on successively hydrothermal and alkaline treatment had a maximum glucose concentration of 18.6 g/L, which was due to the high cellulose concentration and surface area for the substrate, accompanying with removal of large amounts of lignins and hemicelluloses. Conclusions: The present study demonstrated that the order of hydrothermal and alkaline treatment had significant effects on the physicochemical properties and enzymatic saccharification of wheat straw. The one-step process based on successively hydrothermal and alkaline treatment is a simple operating and economical feasible method for the production of glucose, which will be further converted into bioethanol.