To be competitive with common chemical surfactants, the cost of rhamnolipid production must be minimized by selecting suitable substrates and optimizing the fermentation process. With different plant oils as substrates, Pseudomonas aeruginosa TIB-R02 can produce rhamnolipids with different structural characteristics that were confirmed by HPLC/MS analysis. Different rhamnolipids had different performances in interfacial tension. The production of rhamnolipid was greatly enhanced by fermentation optimization with palm oil as substrate. A fermentation-defoaming tandem system was developed to resolve the problems of foaming and medium overflow during scale-up. Finally, the titer of rhamnolipid reached 60 g/l and the yield reached 80 % in a 300 l fermentation-defoaming tandem system. The work reveals the potential for producing high-performance rhamnolipids from renewable resources on a large scale.