In this study, self-assembly regenerated silk fibroin (RSF) nanofibers were prepared and observed by Atomic Force Microscope (AFM). Then RSF films containing nanospheres and nanofibers were prepared and dissolved with poly (L-lactide-co-ε-caprolactone) (PLCL) with a blending ratio of 30/70 in hexafluoro-2-propanol (HFIP). In order to determine whether different nanostructures in the solution influence the morphological, structural, and mechanical properties of the final electrospun materials, flat membranes were prepared and characterized by Scanning Electron Microscope (SEM), Fourier Transform Infrared (FT-IR), and mechanical testing. The secondary structure of as-spun materials with RSF nanofibers were not changed, however, the diameter of electrospun fibers decreased and tensile strength and elongation at breaks increased. Electrospun materials with RSF nanofibers have the potential to be used for skin, cartilage, and blood vessels because of their biocompatibility and improved mechanical properties.