In this paper, we present a novel laser-induced oxidation procedure for in situ formation of nickel oxide nanoporous structures directly onto the nickel surface as a highly sensitive nonenzymatic glucose sensor. The formation of mesoporous nickel oxide is confirmed by field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction. Electrochemical properties of the pristine and laser-induced oxidized nickel (LIO-Ni) films were studied using cyclic voltammetry and electrochemical impedance spectroscopy. The unique three-dimensional mesoporous architecture of the oxide film on the LIO-Ni electrode resulted in a dramatic enhancement in electrochemical reduction/oxidation performance with a 10-fold increase in electrocatalytic activity for nonenzymatic glucose oxidation as compared to the pristine-Ni electrode. The LIO-Ni biosensor performance was successfully examined for the amperometric detection of glucose over a wide concentration range from 5 μM to 1.1 mM with a high linear sensitivity of 5222 μA mM −1 cm −2 . The limit of detection was obtained as low as 3.31 μM with a signal-tonoise ratio of 3. Furthermore, the LIO-Ni electrode showed outstanding long-term stability, reproducibility, and high selectivity in the presence of various interfering agents including uric acid, L-ascorbic acid, acetaminophen, glutamic acid, and citric acid. The demonstrated laser-induced oxidation process can be potentially adapted to the scalable manufacturing of a wide range of other easyto-use and robust metal oxide-based sensors for nonenzymatic biosensing applications.