The invention relates to the technical field of extracting plant sterols, in particular to a method for separating and purifying plant sterols from seabuckthorn seeds. The method specifically comprises the steps of seabuckthorn seed smashing and drying, ultrasonic-assisted leaching and suction filtration, vacuum concentration, saponification, cooling, extraction, water washing, concentration, water bath reflux, recrystallization, washing, centrifugation and drying. Compared with the prior art, the method for separating and purifying the plant sterols from the seabuckthorn seeds has the advantages of simple operation method, low equipment requirement, low cost of reagents, low energy consumption, and high extraction rate of seabuckthorn seed oil, and the highest extraction rate can reach to12.98%, seabuckthorn sterol has a high purity of up to 1.487%, and the method for separating and purifying the plant sterols from the seabuckthorn seeds is suitable for use in small and medium-sizedproduction and processing enterprises in laboratories or production.