The invention relates to a method for preparing a biomaterial of acellular small intestinal submucosa, which comprises the steps of preposition treatment, acellular treatment, enzyme treatment, preparations of membranous products and particle products. Compared with the prior art, the biomaterial has higher bioactivity and biocompatibility, no obvious immune rejection and no toxic effect on cells; besides, the biomaterial has a certain mechanical strength and toughness, and variable shape, size and thickness, thereby being convenient for clinical suturing and fixing. At the same time, the preparation method has the advantages of unlimited raw material source, cell-free residues, no ethical issues and being capable of effectively inactivating virus. The prepared products are applicable to the biomedical engineering fields, such as repairing defections of body tissue, serving as tissue filling materials, repairing facial depression deformity, serving as tissue reinforcements to replace fascia, repairing membranous defections and malnourished and infected surfaces of wound, serving as materials for biodegradable stents, and serving as injectable filling materials.