The invention discloses a self-assembling method for preparing fibroin microspheres. The method comprises the following steps: shearing cocoon shells of silkworms into pieces, putting the sheared cocoon shells into a Na2CO3 water solution to be subjected to degumming treatment, and cleaning with deionized water; putting the cleaned fibroin sample into the Na2CO3 water solution to be subjected to the degumming treatment again, cleaning with deionized water, then flatly spreading the fibroin sample in a baking oven, drying to obtain silkworm fibroin fiber and the like until a milk white liquid is obtained, and then freeze-drying into powder, so that a finished product can be prepared. According to the invention, the fibroin microspheres are prepared without adding any poisonous chemical cross-linking agents, the preparation technology is simple, the reaction condition is moderate, and the fact that the structural characteristics of silk fibroin is kept is facilitated, the utilization ratio of silk fibroin is increased, the production cost is reduced, and the yield of fibroin microspheres is increased. The fibroin microspheres prepared by adopting the self-assembling method can be applied to the fields of medicine carrying micro-system, cellular uptake and the like, and a new thought and a new method are provided for the development of biomedicine.