The invention relates to a method for preparing glucose syrup by performing acidolysis cellulose biomass by a negative pressure mass transfer and diffusion dialysis membrane separation technology. The method is characterized by comprising the following steps of: performing concentrated acid prehydrolysis on the cellulose biomass by a negative pressure cavitation technology with independent intellectual property rights; obtaining acid liquor and the glucose syrup by means of diluted acid high-temperature secondary hydrolysis and diffusion dialysis membrane separation; and concentrating and reusing the acid liquor through negative pressure film formation equipment. In the process, the acid is separated from glucose and is concentrated by means of diffusion dialysis membrane separation equipment and the negative pressure film formation equipment with independent intellectual property rights, and the whole process has the advantage of low energy consumption; the acid liquor is recycled, so that the whole process has the characteristics of no pollution, no waste materials and the like, and the obtained products have the advantages of high conversion rate and high yield. According to the method, a wide prospect is opened for expending raw materials for preparing the glucose, and utilizing the cellulose biomass at high added value.