The invention discloses a pretreatment method for detecting the content of 2'-fucosyllactose in milk powder, and belongs to the technical field of food detection. The method comprises the following steps: dissolving, centrifuging, diluting and membrane passing-through. In the dissolving step, a precipitator is used for dissolving the milk powder sample; a precipitant is one of methanol, ethanol, acetonitrile and a buffer solution. Compared with other pretreatment methods, the method is simple and convenient to operate, high in detection sensitivity, high in separation degree and the like, interference of lactose is greatly reduced, the quantification limit of 2'-fucosyllactose is improved, the quantification limit is as low as 100 [mu]g/g, and the method has important significance in detection accuracy of low-content 2'-fucosyllactose.