Cobalt oxide-doped copper oxide composite nanofibers (CCNFs) were successfully achieved via electrospinning followed by thermal treatment processes and then exploited as active electrode material for direct enzyme-free fructose detection. The morphology and the structure of as-prepared samples were investigated by X-ray diffraction spectrum (XRD) and scanning electron microscopy (SEM). The electrocatalytic activity of CCNFs films towards fructose oxidation and sensing performances were evaluated by conventional electrochemical techniques. Cyclic voltammetry (CV) and chronoamperometry (I−t) revealed the distinctly enhanced sensing properties towards fructose compared to pure copper oxide nanofibers (CNFs), i.e., showing significantly lowered overpotential of 0.30V, ultrafast (1s) and ultrasensitive (18.988μAmM−1) current response in a wide linear range of 1.0×10−5 M to 6.0×10−3 M with satisfied reproducibility and stability, which could be ascribed to the synergic catalytic effect of the binary CuO/Co3O4 composite nanofibers and the highly porous three-dimensional network films structure of the CCNFs. In addition, a good selectivity for fructose detection was achieved. Results in this work demonstrated that CCNFs is one of the promising catalytic electrode materials for enzymeless fructose sensor fabrication.