Co₃O₄ microspheres with free-standing or bundled nanofibers (NFs) were fabricated for use as a platform for non-enzymatic glucose sensing. The sensor based on free-standing Co₃O₄ NFs displays enhanced sensitivity (1440 μA mM⁻¹ cm⁻²), a wider linear range (0.005–12 mM) and superior selectivity. The application of this glucose sensor in human blood serum has also been demonstrated successfully.