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The effects of sulfur trioxide micro-thermal explosion (STEX) and enzyme loading on reducing sugars conversion of STEX-treated rice straw and enzymatic hydrolysates were researched. Important process parameters in the pretreatment of biomass were identified by a Plackett-Burman design, and parameters with significant effects were optimized using a Box-Behnken design (BBD) and response surface methodology (RSM). The optimal conditions were a temperature of 80 °C and a treatment time of 30 min when only single factors were considered. Meanwhile, glucose and xylose were primary components in the enzymatic hydrolysates. Subsequently, STEX time, liquid-solid ratio, and soaking temperature were the main factors governing the enzymatic saccharification of rice straw. The optimum pretreatment conditions were STEX time 23.3 min, liquid-solid ratio 13.3 (V/m), and soaking temperature 62.2 °C. The chemical composition analysis of straw further demonstrated that STEX collaborative dilute lye pretreatment could remove lignin and hemicellulose.