The tussah silk fibroin (TSF)/poly(lactic acid) (PLA) composite nanofibers with different composition ratios were prepared by electrospinning with 1,1,1,3,3,3-Hexafluoro-2-propanol as the solvent. The morphology and secondary structure of the fibers were characterized by Scanning electronic microscope, Fourier transform infrared (FTIR), and X-ray diffraction (XRD). The thermal and mechanical tests were also performed. The spinnability of TSF solution was improved significantly through adding 10% PLA, and the average diameter of the fibers decreased from 583 nm to 178 nm with an obvious improvement in fiber diameter uniformity. In addition, the mechanical properties of electrospun nanofibers increased evidently after blending 10% PLA, whereas the thermal properties kept stable. FTIR and XRD analysis indicated the addition of 5% PLA could induce a conformation transformation of TSF from random coil and alpha-helix to beta-sheet, however, when PLA content was more than 10%, the beta-sheet structure of TSF in composite nanofibers decreased, and the phase separation of two compositions occurred. Therefore, when PLA content exceeded 15%, the average diameters of TSF/PLA composite nanofibers increased and appeared to be polarized, moreover, the mechanical properties of the fibers decreased with the increase of PLA content, and the fibers displayed the mechanical behavior of PLA component more.