The present invention discloses a preparation method of a soft tissue decellular matrix, and relates to the technical field of clinical medicine, biomedical medicine and regenerative medicine. According to the present invention, the natural soft tissue decellular matrix is prepared by using a supercritical fluid technology, wherein the natural soft tissue comprises small intestine tissue, blood vessel tissue, muscle tissue, heart tissue, valve tissue, lung tissue, spleen tissue, kidney tissue, liver tissue, stomach tissue, gallbladder tissue, fat tissue, cartilage tissue, trachea tissue, esophagus tissue, bladder tissue, ureter tissue, oviduct tissue, or uterus tissue. According to the present invention, with the preparation method, the damage on the soft tissue matrix component is low, and the obtained soft tissue decellular matrix has advantages of low immunogenicity and good biocompatibility.