Microcomposite fibers of regenerated silk fibroin (RSF) and multiwalled carbon nanotubes (MWNTs) were successfully prepared by an electrospinning process from aqueous solutions. A quiescent blended solution and a three-dimensional Raman image of the composite fibers showed that functionalized MWNTs (F-MWNTs) were well dispersed in the solutions and the RSF fibers, respectively. Raman spectra and wide-angle X-ray diffraction (WAXD) patterns of RSF/F-MWNT electrospun fibers indicated that the composite fibers had higher beta-sheet content and crystallinity than the pure RSF electrospun fibers, respectively. The mechanical properties of the RSF electrospun fibers were improved drastically by incorporating F-MWNTs. Compared with the pure RSF electrospun fibers, the composite fibers with 1.0 wt % F-MWNTs exhibited a 2.8-fold increase in breaking strength, a 4.4-fold increase in Young's modulus, and a 2.1-fold increase in breaking energy. Cytotoxicity test preliminarily demonstrated that the electrospun fiber mats have good biocompatibility for tissue engineering scaffolds.