The invention relates to an acellular biological patch, a preparation method and an apparatus thereof. In the invention, small intestines of pigs are taken as raw materials, which are subjected to a series of decellularization treatment by means of an acid, hydrogen peroxide, a mixed solution of high salt and alkali so as to obtain the acellular biological patch. According to the preparation method, after treatment by the hydrogen peroxide, the mixed solution of high salt and alkali can be employed to conduct twice recycling treatment, thus reaching the effects of thorough decellularization and antigen removal. The employment of a transverse and longitudinal alternating laying way maintains uniformity of the patch's mechanical properties, the tensile strength, suture tear force, tear strength and other mechanical indexes can well meet clinical demands, thus facilitating surgical suture operation. The employed drying mode has no need of any adhesive and suture line for fixation, so that a natural three-dimensional support structure of the product is maintained, and after implantation into the human body, no strong inflammatory response and immunological rejection response can occur. The patch has no toxicity on cells, so that infection and adhesion cannot occur in a repair process. The acellular biological patch provided in the invention is suitable for repair of body tissue defects and soft tissue injuries, and also can be used in the bioengineering field to serve as the tissue for reinforcing repair of membranous defects and infected wounds and serve as a biological scaffold material, etc.