The invention discloses a preparation method of silk fibroin nanospheres, which comprises the following steps: dispersing a silk fibroin solution into nanometer liquid drops by an inverse emulsion method, adding polyols under ultrasonic oscillation to promote silk fibroin in the nanometer liquid drops to form a structure insoluble in water, adding high water-absorbing resins to dry and cure the silk fibroin in a microemulsion state so as to obtain silk fibroin nanospheres which are insoluble in water. The silk fibroin nanospheres have good biocompatibility, are nontoxic and harmless for human body, and have no immunogenicity; the preparation process adopts mild technology with a normal temperature, a normal pressure, and no chemical cross-linking agents; therefore, the product can be used as a carrier with biologically-active substances to load enzymes, nucleic acids, polypeptides, protein drugs, and the like; the obtained silk fibroin spheres have small particle sizes, good dispersibility, do not adhere, and can be prepared into various cosmetics, skin care products, sunscreen creams and the like; the silk fibroin spheres have small particles which can run freely in blood, thus can be used as injections and have wide application in fields of disease diagnosis and treatment.