The invention provides a preparation method of a root system anti-freezing solution. The preparation method comprises the following steps: preparing the following materials: paraffin, calcium chloride, methanol, fulvic acid, glycerol, distilled water, mono carboxylic acid, ramulus mori, pine needle, rhizoma zingiberis and herb of diffuse dayflower; A, mixing methanol, glycerol, distilled water and mono carboxylic acid, uniformly stirring, then adding fulvic acid into the mixture, adding paraffin (liquid paraffin) into the mixture, uniformly stirring and leaving to stand; B, placing ramulus mori, pine needle, rhizoma zingiberis and herb of diffuse dayflower into a closed vessel, adding an ethanol solution with the mass percent of 75 into the closed vessel, infiltrating and soaking the active components for 12 h, after that, charging the active components into a percolator, adding ethanol with the mass percent of 75 for soaking, then starting to collect the percolating liquid, heating the collected percolating liquid with 75 percent of ethanol to reclaim ethanol so as to obtain an extract, adding the extract into calcium chloride to enable the extract to be dissolved, heating dissolved calcium chloride to be 40-50 DEG C, and sterilizing for later use; C, fully mixing the product obtained in the step A and the product obtained in the step B. The proportioning is simple, the use is convenient, frost injury loss of trees is greatly reduced and the anti-freezing effect is good.