Waste crude oil emulsion was inevitably produced in the petroleum industrial process, causing harmful impact on the ecological and social environment. In this study, rhamnolipid was for the first time investigated for demulsification of waste crude oil. As found in this paper, rhamnolipid treatment could obtain over 90% of dewatering efficiency on refractory waste crude oil and such efficient demulsification was confirmed on model emulsions. As further demonstrated on the pilot scale (100L), rhamnolipid treatment could recover over 98% of crude oil from the wastes. The recovered oil contained less than 0.3% of water and thus can directly re-enter into refinery process while the aqueous phase can be disposed into dischargeable water due to largely reduced soluble COD after subjected to 5days of active sludge treatment. It seems that rhamnolipids as biodemulsifiers were of great prospects in the industrial demulsification of waste crude oil.