The invention relates to a preparation method of a silk fibroin micro-nanofiber porous scaffold. The preparation method comprises the steps that acid enters silk to enable silk fibers to generate full swelling; freezing-thawing treatment is conducted multiple times to enable fibrils in the silk fibers to be split; the split silk fibroin micro-nanofiber porous scaffold is obtained through a freeze drying technology. The preparation method is simple and easy to operate; the interior of the silk fibroin micro-nanofiber porous scaffold prepared through the method is mainly composed of natural silk fibroin fibers with the diameters ranging from 10 nanometers to 10 micrometers, the silk fibroin micro-nanofiber porous scaffold has the good chemical stability and physical and mechanical properties and the bionic micro-nano structure, and therefore the silk fibroin micro-nanofiber porous scaffold is very suitable for serving as a regenerative medicine scaffold material for cell and tissue growth.