In this work, rhamnolipid production was investigated using waste frying oil as the sole carbon source. By culture in shaking flasks, a naturally isolated strain synthesized rhamnolipid at concentration of 12.47 g/L and its mutant after treatment by UV light increased this productivity to 24.61 g/L. Fermentation was also conducted in a 50 L bioreactor and the productivity reached over 20 g/L. Hence, with a stable and high productive mutant strain, it could be feasible to reuse waste frying oil for rhamnolipid production on industrial scale.