Cellulose nanocrystals-reinforced chitosan-poly(ethylene oxide) (CNC/CS-PEO) nanofibers were successfully prepared by electrospinning. The nanofibers reinforced with CNC exhibited excellent mechanical performance. The tensile strength of CNC/CS-PEO nanofibers with 6 wt% CNC loading was increased by 5.76 MPa, 33.5% higher than that of CS-PEO nanofibers. Smooth nanofibers with diameters of about 500 nm could be obtained with lower CNC loading. The diameter of CNC/CS-PEO nanofibers increased with increase in CNC loading and cross-linking between fibers was observed with 8 wt% CNC loading. The thermal stability of nanofibers was enhanced due to interactions between CS-PEO matrix and CNC nano-reinforced phase. Too strong or too weak CNC chain migrations reduced the crystallization of CS-PEO. FT-IR results showed that CS-PEO and CNC were physically mixed together. The CNC reinforced nanofibers show excellent performance, have low cost, and are degradable and hence have potential applications in biological field.