ABSTRACT The wide expansion of the meat industry generated tremendous amounts of animal-based waste. Discarded beef tallow (DBT) is one of those prevalent lipid-rich wastes disposed of by slaughterhouses, meat processing units, and tanneries. The current study utilized the advanced technology of supercritical CO2 (SC-CO2) to extract lipid content from DBT for biodiesel production through transesterification. The effects of SC-CO2 parameters on the extraction rate were investigated over ranges of (32–80°C) for temperature, (10–50 MPa) for pressure, and (15–150 min) for treatment time. Using response surface methodology (RSM), both processes of SC-CO2 and transesterification were optimized. The highest extraction rate was 86.10%, obtained at the experimental conditions of 60°C for temperature, 30 MPa for pressure, and 120 min for treatment time. For the transesterification, the maximum yield of biodiesel was 95.15%, obtained at the reaction conditions of 1:7.5, 1.16 wt%, 58°C, and 72 min for lipid to methanol molar ratio, catalyst ratio, temperature, and time, respectively. The outcomes of the kinetic and thermodynamic analyses showed that the SC-CO2 extraction was an endothermal, unspontaneous, and temperature-dependent process. The characteristics of the synthesized biodiesel largely comply with the ASTM D6751 and EN 14,214 standards. The findings of the current study confirm the viability of SC-CO2 to extract lipids from DBT as a low-cost feedstock for biodiesel production.