This article proposes for consideration the experimental results of applying the method of laser surface modification for the manufacture of a highly sensitive enzyme-free sensitive layer of the working electrode of a glucose sensor using nickel oxide as an example. This technology can be integrated into the lab-on-a-chip concept. Using cyclic voltammetry, the sensitivity of the proposed system to the addition of microdoses of glucose to the solution for unmodified and laser-modified nickel surfaces was studied. The formed structure of metal oxide sensors was studied for the determination of D-(+)-glucose in the range of analyte concentrations up to 1.5 µM. In addition, the sensitive layers made using the proposed technology showed durability and reproducibility of properties. This allows us to conclude that the demonstrated method of laser-induced metal surface modification is suitable for the production of a wide range of enzyme-free glucose sensors.