In this study, regenerated silk fibroin (RSF, from Bombyx mori) nanofibers with smooth surface had been successfully prepared via electrospinning, as shown by SEM and then as-spun fibers were induced under 75% ethanol vapor. We aimed to investigate the morphology and structure change of 75% ethanol vapor-induced silk fibroin nanofibers. To determine any difference in surface topographies, the nanofibers were inspected using atomic force microscope (AFM) and the results showed that after inducement of 75% ethanol vapor for 24 h, the surface of fibers became rough. Differential Scanning Calorimetry (DSC) analysis indicated that electrospun SF nanofibrous membranes typically took silk I form and 75% ethanol vapor-induced SF nanofibrous membranes took silk II structure. These results suggested that 75% ethanol vapor inducement could be an attractive alternative to expand the application of RSF.