The invention discloses a high-throughput preparation method of multicellular tumor spheroids. The preparation method comprises the following steps: S1, quickly mixing a cell suspension of tumor cellswith a basement membrane matrix, and transferring the obtained cell suspension into a low-attachment well plate; and S2, performing centrifuging, culturing the cell suspension in an incubator after centrifugation, and maintaining the cell suspension during culture to form the multicellular tumor spheroids combined with the basement membrane matrix. The added substance used in the technical schemeis the basement membrane matrix Matrigel which can promote the formation and growth of the multicellular tumor spheroids, thereby promoting the three-dimensional structure of the formed multicellulartumor spheroids; meanwhile, the low-attachment well plate is used, so the tumor cells spontaneously aggregate into spheroids; and combined with culture after centrifugation of the well plate and thedesign of the step of maintaining the cell suspension during culture, cell aggregation is artificially promoted by centrifugation, so the subsequently formed cell spheroids are more compact and have amore three-dimensional structure. Therefore, the prepared tumor spheroids are in a state of uniform diameter and concentrated particle size distribution.