This research was investigated to enhance the efficiency of enzymatic hydrolysis on wheat straw via freeze-thawing pretreatment, and to find the physicochemical structural changes after this pretreatment. Results show that, this pretreatment enhances the enzymatic hydrolysis efficiency of cellulose and hemicellulose, and hemicellulose is more susceptible to this pretreatment. The highest enzymatic hydrolysis efficiency of cellulose and hemicellulose was 57.06% and 70.66% occurred on -80℃, 24 h and -10℃, 24 h pretreated samples, with an improvement of 2.23 and 3.13 folds of control. SEM images exhibited that transverse cracks appeared before longitudinal cracks as pretreated conditions aggravated, and holes can be found on every sample after this pretreatment. FTIR and XRD analysis implied that freeze-thawing pretreatment had effect both on the crystalline and amorphous regions, and disrupted the hydrogen bonds among them. This study provided a physical pretreatment method to improve the efficiency of enzymatic hydrolysis on wheat straw.