Abstract: To address the issues of enzyme-based lactate sensors, including enzyme deactivation, high fabrication cost, and limited capability for on-site rapid detection of sweat lactate, this study constructs a PDMS-based flexible three-electrode system using screen printing. A NiO-Nafion composite catalytic layer was fabricated on the working electrode via drop-casting, and a disposable flexible NiO-Nafion enzyme-free sensing electrode was developed. The interfacial electrochemical behavior was characterized by cyclic voltammetry （CV）, chronoamperometry （IT）, and electrochemical impedance spectroscopy （EIS）. The effects of NiO loading and applied potential on the sensing performance were systematically investigated. The results show that under the optimal conditions of NiO loading of 150 μg and applied potential of 0.45 V, the electrode exhibits a detection range of 0.1~30 mM, fully covering the physiological concentration range of human sweat. Two linear segments were observed in the ranges of 0.1~8 mM and 8~30 mM, with sensitivities of 57.1 and 24.05 μA·mM⁻¹·cm⁻², respectively. The limit of detection was as low as 18 μM, with a response time of approximately 30 s. Furthermore, the electrode exhibits excellent bending resistance and good batch-to-batch reproducibility. The detection accuracy in complex matrices was verified through artificial sweat calibration and ex vivo sweat sample testing. This study demonstrates that the disposable flexible enzyme-free electrode combines low cost with high sensing performance, showing potential application value in portable point-of-care detection and exercise physiology assessment.