The invention relates to a preparation method of a decellularized anti-calcification heart patch. The method mainly comprises the steps that raw material pericardial tissue is degreased through an organic reagent and processed by a mixed solution of a high salt, a surface active agent and alkali, and finally sterilization treatment is performed to obtain the decellularized anti-calcification heart patch. According to the obtained heart patch, due to the special decellularized anti-calcification technology, the collagenous fiber three-dimensional pore structure of the raw materials is reserved, meanwhile, cells contained in the materials are effectively removed, and the immunogenicity is lowered; the materials can guide cells to grow in the materials, scar tissue generation is reduced, and the good anti-calcification ability is achieved; the excellent mechanical property is achieved, arterio-venous pressure difference between heart chambers can be resisted, and the repair effect is ensured. The preparation method is applicable to atrial septal defects, ventricular septal defects, aortic stenosis and the like caused by the congenital heart disease.