The invention provides a pine pollen extract for treating fatty liver as well as a preparation method and an application thereof, relating to the technical field of extracting effective substances from gymnosperm pine pollen. The preparation method comprises the following steps: the pine pollen is subjected to wall breaking at a low temperature, and impurities in the pine pollen are removed; the pine pollen is extracted with ultrasonic after the pine pollen is immersed in normal-pressure normal-temperature water; solid impurities are centrifugally removed to obtain an aqueous solution; and a gymnosperm pine pollen aqueous extract with the molecular weight lower than 3,000D is separated at the environmental temperature of between 1 and 5 DEG C to obtain the gymnosperm pine pollen aqueous extract for treating the fatty liver. The pine pollen aqueous extract can be used for general health-care for children, and can prevent occurrence of fatty liver. The pine pollen aqueous extract can be used for preparing an oral preparation for treating the fatty liver, has a good absorption effect and low price, and also can be used for preparing an injection preparation for treating the fatty liver, which can be absorbed quickly, has strong treatment pertinence.