The invention discloses a rooting agent for pine cuttings, which includes a root dipping agent and a spraying agent. The root dipping agent mainly includes the following raw material solutions: dihydrogen phosphate, magnesium nitrate hexahydrate, boric acid, nicotinic acid, biotin, Inositol, Phloroglucinol, ABT 1 , melatonin, methylpiperium, abscisic acid; the spraying agent mainly includes the following raw material solutions: choline chloride, gibberellin, mancozeb, streptomycin. The invention also discloses a preparation method and a use method of the above-mentioned pine cutting rooting agent. The rooting agent prepared by the invention has strong rooting ability, can promote the rooting of pine cuttings, improve the efficiency and quality of rooting of pine cuttings, and can inhibit the growth of mold during the rooting process and prevent the cuttings from mildew.