Detection and monitoring of lactate and glucose levels in biological fluids and cell cultures are essential for understanding metabolic disorders. While electrochemical biosensors are commonly used, traditional enzymatic sensors face challenges related to stability, reproducibility, and cost. To address these limitations, we developed non-enzymatic sensors for lactate and glucose detection using nanostructured nickel oxide (NiO)–modified screen-printed carbon electrodes. The sensors were fabricated by drop-casting a NiO/Nafion/ethanol dispersion onto the working electrode, and their performance was evaluated using cyclic voltammetry and amperometry. Optimal sensitivity and linearity were achieved at a working potential of ∼0.5 V. The sensors exhibited linear responses for both lactate and glucose in the 0.1–5 mM range, with detection limits of 0.03 mM (lactate) and 0.025 mM (glucose), and sensitivities of 1.564 μA/mM (lactate) and 1.842 μA/mM (glucose) in 0.1 M NaOH–KCl electrolyte. To address glucose interference in lactate sensing, dual-sensing strategies were employed by varying Nafion concentration, applying differential potentials, or modifying the sensors with Prussian Blue to achieve selective detection. Validation against commercial lactate and glucose assay kits in cell culture medium showed good agreement, confirming the sensors’ accuracy. Finally, the sensor was integrated with a microfluidic chip, demonstrating its potential as a flow-through, enzyme-free metabolic sensor for future organ-on-a-chip applications.