The rise in prices of fossil fuels, environmental constraints associated with climate change, as well as geopolitical conditions, are driving the search for alternative energy sources in the 21st century. The use of used fried oil (UFO) as an economical resource constitutes a viable and technically feasible strategy—at both the meso and macro levels—for R&D programs at universities in Ecuador and for the implementation of policies aimed at utilizing high-value-added waste products. UFO from the city of Esmeraldas, Ecuador, was used as a low-cost feedstock to produce biofuel (biodiesel) via transesterification under heterogeneous catalytic conditions. This UFO was filtered through cellulose to reduce the acidity and remove suspended solids. Vanadyl phosphate (VOPO₄), a solid heterogeneous catalyst, was used in the process; it was synthesized from spent divanadium pentoxide derived from sulfuric acid synthesis. The preparation of this catalyst involved combustion in microwave ovens to remove sulfur gases and sonochemical synthesis with phosphoric acid under ultrasound. The biodiesel production process was carried out in a mini-reactor, with operating conditions set at 115 °C and 80 PSI. The optimal operating conditions were determined: the addition of 3% VOPO₄ catalyst relative to the weight of the oil, an oil-to-methanol molar ratio of 1:12.83, and a reaction time of 4.85 hours. Operating under these parameters, the biodiesel production yield exceeded 80% per batch. The quality and composition of the biodiesel produced were validated using gas chromatography, which demonstrated the formation of fatty acid methyl esters, such as methyl palmitate, stearate, oleate, and linoleate. Infrared (IR) spectroscopy revealed a strong analytical signal at 1740 cm⁻¹, which is characteristic of the ester functional group (C(O)-O-). Final testing demonstrated that properties such as density, viscosity, flash point, and sulfur content of the biofuel satisfactorily meet the rigorous technical quality standards and requirements established by ASTM and INEN (Ecuadorian) standards.