The invention discloses a method for processing and preparing resistant starch by Pullulanase cooperated with acid alcohol, comprising the following steps: heating starch milk of which the weight concentration is 10-45% and the pH value is 3.5-6.5 to 40-70 DEG C, adding Pullulanase of which the dosage is 1-40ASPU per gram of starch, and keeping for 8-48hrs; inactivating enzymes, assisting by acid alcohol processing, cooling, filtering, drying, crushing and sieving to obtain the product. The invention takes starch as a raw material, adopts the biological technology and acid alcohol processing means, thus greatly improving the content of the resistant starch, simplifying the process for preparing the resistant starch, effectively enhancing the yield, and lowering energy consumption. The obtained product not only has the function of dietary fiber, but also has the characteristics of fat substitute, and can be widely used as functional food materials and biochemical drug carriers.