This study reports the performance of simple low-cost synthesized bifunctional Cu/Cu2O nanoparticles (NPs) used as a catalyst for energy-harvesting applications through of a microfluidic fuel cell (µFC), and further, as cholesterol (Chol) sensor. TEM characterization of the NPs showed spheres between 4 and 10 nm, while XRD and XPS analysis confirmed the composition and preferential crystallographic plane of Cu/Cu2O. In addition, 25.26 m2 g−1 surface area was obtained, which is greater than those commercial materials. NPs showed high activity toward the cholesterol oxidation reaction when were used as a sensor, obtaining a linear interval between 0.5 and 1 mM and 850 µA mM−1 mg−1 of sensitivity and 8.9 µM limit of quantification LOQ. These values are comparable to results previously reported. Moreover, Cu/Cu2O NPs were used as anode in a µFC with 0.96 V of cell voltage and 6.5 mA cm−2 and 1.03 mW cm−2 of current and power density, respectively. This performance is the highest currently reported for cholesterol application as an alternative fuel, and the first one reported for a microfluidic fuel cell system as far as is known. Results showed that the obtained Cu-based NPs presented an excellent performance for the dual application both µFC and sensor, which has potential applications in biomedicine and as an alternative energy source.