© People with diabetes require regular blood sugar level monitoring, using commercial enzyme-based biosensors. There is a considerable need to develop biosensors with nonenzymatic electrodes to eliminate the drawbacks of enzymes. Nanostructured nickel oxide (NiO) thin films are highly promising materials for the development of nonenzymatic glucose and hydrogen peroxide (H2O2) biosensors. Although the biosensor performance can be easily attained with nonenzymatic electrodes, their commercialization still requires development of cost-effective and mass-production methods. In this work, we demonstrate the use of ultrasonic spray deposited, nanometer-thick, manganese and cobalt doped NiO (Mn:NiO and Co:NiO) films on indium tin oxide (ITO) coated glass substrates for glucose sensing. Sensor characterization followed detailed materials characterization. Nanometer-thick Co:NiO film electrodes showed better glucose sensor performance than those of bare NiO and Mn:NiO electrodes. High sensitivity of 1.67 μA/μM·cm2, a low detection limit of 231 nM, and a fast response time of 5.4 s within the linear range of 16-308 μM were obtained from nanometer-thick Co:NiO film electrodes. Amperometric measurements showed significant electrode reproducibility and stability. Nanometer-thick Co:NiO film electrode was also used to demonstrate actual clinical glucose measurements using human blood serum as a glucose source. Moreover, all fabricated nanometer-thick film electrodes were also utilized as H2O2 sensors. This work provides a novel approach for monitoring the biosensor performance using nanometer-thick doped NiO film electrodes. Obtained results demonstrated the potential of ultrasonic spray deposition method for the mass-production of high-performance nonenzymatic nanometer-thick film biosensors.