The tussah silk fibroin (TSF) nanofibers with 611 nm diameters were prepared by electrospinning with the solvent hexafluoroisopropanol (HFIP). And then, the TSF nanofibers were crosslinked by 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide/ N-Hydroxysuccinimide (EDC/NHS) crosslinking agent. The morphology and microstructure of the crosslinked TSF nanofibers were characterized by scanning electron microscopy (SEM), Fourier transforms infrared analysis (FTIR), X-ray diffraction, Instron electronic strength tester, and cell culture. After treatment with EDC/NHS crosslinking agent, the TSF nanofibers swelled and its average diameter increased from 611 to 841 nm. FTIR and X-ray diffraction results demonstrated that random coil, ${alpha}$-helix, and ${eta}$-sheet co-existed in the TSF nanofiber mats, but the content of ${eta}$-sheet increased from 25.26 to 45.34 %, and the random coil content decreased from 32.47 to 24.94 %. Compared with the electrospun pure TSF nanofiber mats, the crosslinked TSF nanofiber mats exhibited a lower breaking tenacity and initial modulus, which were 5.51 MPa and 9.86 MPa, respectively. At the same time, the extension at break of the crosslinked TSF nanofiber achieved 109.38 %. In cell culture evaluation, the crosslinked TSF nanofibers were found to support cell adhesion and spreading fibroblast L373 and bone marrow mesenchymal stem cells (BMSCs), which had potential utility in a range of tissue engineering.