Abstract: In order to improve the spinnability of chitosan (CS)-silk fibroin (SF) composite nanofibers and solve the problem of poor dimensional stability of nanofiber membrane caused by its easy swelling, polyethylene oxide (PEO) was used to improve the spinnability of the composite fibers in this study. Electrospinning was used to prepare CS-PEO-SF nanofibers, which were cross-linked and by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). The intermolecular interaction, crystallization properties, mechanical properties and morphology of CS-PEO-SF nanofibers were characterized by FTIR, XRD, single fiber strength machine and SEM. The swelling degree and biocompatibility of CS-PEO-SF nanofibers was also characterized by cytotoxicity and cell culture tests. The results show that the addition of PEO can effectively improve the spinnability of CS-SF. The average diameter of CS-PEO-SF nanofibers is from 240 nm to 510 nm. The fiber morphology of each component is good. With the increase of SF content, the crystalline properties, tensile strength and breaking elongation of CS-PEO-SF nanofibers gradually increase. After crosslinking, the crystallinity and swelling degree of the CS-PEO-SF nanofibers decrease, and the mechanical properties increase. Before and after cross-linking, the CS-PEO-SF nanofibers has no toxicity and has good cell compatibility.