The invention discloses a silk fibroin nanofiltration film with the surface with a micro-nano structure as well as a preparation method and application of the silk fibroin nanofiltration film. The preparation method comprises the following specific operation steps: uniformly mixing a silk fibroin solution obtained from silk through degumming, dissolving, dialysis, filtration, centrifugation and concentration with a polymer solution, and carrying out electrostatic spinning and after-treatment, so as to prepare the silk fibroin nanofiltration film with the surface with the micro-nano structure, wherein the diameters of nanofibers are 100nm-1000nm, the thickness of the film is 2-10 microns, the porosity of a fiber film is 60%-85%, the pore size is 20nm-500nm, and the filtration efficiencies of the film to PM2.5 and PM10 can reach above 99%. The preparation method is simple in process, the process is non-toxic and environment-friendly, and the silk fibroin nanofiltration film has wide application prospects in the fields of personal and industrial air filtration equipment.