The invention provides a preparation method of a decellularized biological amnion. The preparation method comprises cleaning, chemical treatment and freeze drying of the amnion. According to the method, a fresh amnion is washed clean with normal saline, residual blood on the amnion is washed away, the obtained amnion is treated with hydrogen peroxide, acetone, sodium dodecyl sulfate and alcohol again, after alcohol treatment, the obtained amnion is subjected to profound hypothermia freezing in an environment at subzero 70 DEG C for 16-25 h, and the obtained amnion is placed in a freezer dryer for freeze drying according to a set freeze drying curve. A finished decellularized biological amnion is obtained after irradiation sterilization. The amnion is jointly treated with hydrogen peroxide, acetone, sodium dodecyl sulfate and alcohol, immunogenic ingredients of an epithelial cell layer, a fibrous membrane cell layer and a sponge layer in the amnion can be effectively removed, meanwhile, a basilar membrane and a compact layer of the amnion are reserved, and the biological activity and biomechanical properties of the amnion are better reserved; on the other hand, the immunogenicity of the decellularized biological amnion can be further reduced through freeze drying treatment, and the immunological rejection of host cells is reduced. In addition, the preparation method of the decellularized biological amnion is simple and easy to implement, and large-scale production operation is facilitated.