The invention discloses a preparation method of in-situ silk fibroin-povidone iodine composite hydrogel. The method comprises the following steps: dissolving silk fibroin lyophilized powder into deionized water, and fully shaking to obtain a silk fibroin aqueous solution with the concentration of 5 to 30 percent; extracting a certain amount of medical povidone iodine according to the volume ratioof the silk fibroin aqueous solution to a povidone iodine solution being 1:3-1:5, and respectively storing a silk fibroin deionized aqueous solution and the extracted medical povidone iodine solutionin injectors; docking the two injectors by a double-ended Luer taper; carrying out multi-time mutual repulsion on the solution to form a blended solution; solidifying the blended solution for 2 to 8 hours to form the in-situ silk fibroin-povidone iodine composite in-situ hydrogel.