In this study, the fermentable sugar released from cotton stalk (CS), which were pretreated by instant catapult steam explosion (SE) combined with different concentrations of strong monobasic acid (HCl), weak monobasic acid (CH3COOH), strong monobasic alkali (NaOH) and weak monobasic alkali (NH3 center dot H2O), followed by hydrolysis in cellulase/xylanase mixed enzyme solutions, were comparably investigated. The highest yield of 73.22% of fermentable sugar yield was obtained in SE-2.4 MPa-5% NH3 center dot H2O treated CS substrates, which was 5.14 times higher than that from enzymatic hydrolysis (EH) of raw CS. Furthermore, evaluation of monolignins content (H, G, S) in different CS samples suggested that substrates rich in guaiacyl (G) and syringyl (S) would generate a higher efficiency of enzymatic saccharification. Therefore, the slight genetic modification of monolignins for cotton stalk might be a potential way to enhance biomass degradation and transformation.