The invention provides a biochemical joint preparation method and application of absolute rose oil. The preparation method comprises the steps that coarse rose powder is prefermented by beta-glucosidase, amylase and cellulase to generate aroma, reflux extraction is conducted for 3-5 h with an aqueous solution the pH value is regulated to be 9-14, a Maillard reaction is conducted for 3-5 h, suction filtration is conducted, filtrate is retained, an ethanol solution is added into filter residues for secondary reflux extraction, suction filtration is conducted, filtrate is retained, the filtrates obtained in two times of suction filtration are mixed, concentration and alcohol precipitation are sequentially conducted, and a rose extract is obtained; the rose extract is purified through molecular distillation, light components are retained, heavy components are purified through molecular distillation again, the light components are mixed, and the purified absolute rose oil is obtained. The absolute rose oil has purer and richer aroma and fewer impurities, and the irritation is reduced. When the absolute rose oil is applied to reconstituted tobaccos, the color and luster of the reconstituted tobaccos is more natural and uniform, the aroma can be enriched, irritation and offensive odor can be reduced, and the sensory quality can be improved; when the absolute rose oil is applied to a novel tobacco product atomizing agent, the aroma is enriched, the noble rose note is improved, and the tobacco aroma is increased.