A flexible and portable triboelectric nanogenerator (TENG) based on electrospun polyvinylidene fluoride (PVDF) doped with copper oxide (CuO) nanoparticles (NPs, 2, 4, 6, 8, and 10 wt.-% w.r.t. PVDF content) was fabricated. The structural and crystalline properties of the as-prepared PVDF-CuO composite membranes were characterized using SEM, FTIR, and XRD. To fabricate the TENG device, the PVDF-CuO was considered a tribo-negative film and the polyurethane (PU) a counter-positive film. The output voltage of the TENG was analyzed using a custom-made dynamic pressure setup, under a constant load of 1.0 kgf and 1.0 Hz frequency. The neat PVDF/PU showed only 1.7 V, which further increased up to 7.5 V when increasing the CuO contents from 2 to 8 wt.-%. A decrease in output voltage to 3.9 V was observed for 10 wt.-% CuO. Based on the above results, further measurements were carried out using the optimal sample (8 wt.-% CuO). Its output voltage performance was evaluated as a function of varying load (1 to 3 kgf) and frequency (0.1 to 1.0 Hz) conditions. Finally, the optimized device was demonstrated in real-time wearable sensor applications, such as human motion and health-monitoring applications (respiration and heart rate).