In this study, an electrochemical non‐enzymatic glucose sensor based on cobalt oxide was designed through CO2 laser irradiation on a Co‐hydrogel formed by spin‐coating a mixture of CoCl2 and gelatin on a polyimide (PI) film. The PI film, which absorbed IR energy through laser irradiation, was grown into laser‐induced graphene fiber (LIGF) with high electrical conductivity and a large surface area. The Co‐hydrogel absorbed the laser‘s IR energy and grew into laser‐induced Co3O4 nano particles (LICONPs) without high temperature annealing for several hours. Because the Co‐hydrogel and the PI film were simultaneously irradiated with laser, LICONPs were attached to the surface of the LIGF, so it had an active surface area 10.5 times larger than the geometric area. The fabricated LICONPs/LIGF was then utilized as a non‐enzymatic glucose sensor working electrode, resulting in high sensitivity of 187,129 μA mM−1 cm−2 in the range of 1–30 μm and low limit of detection (LOD) of 0.10 μm. In addition, LICONPs/LIGF also showed stable reproducibility, repeatability and long‐term stability when used as a part of working electrode of a non‐enzymatic glucose sensor.