To improve rhamnolipid production and its potential application in removal of crude oil, the recombinant Pseudomonas aeruginosa strain DAB was constructed to enhance yield of rhamnolipids. Strain DAB had a higher yield of 17.3 g rhamnolipids l(-1) in the removal process with crude oil as the sole carbon source than 10 g rhamnolipids l(-1) of wild-type strain DN1, where 1% crude oil was degraded more than 95% after 14 days cultivation. These rhamnolipids reduced the surface tension of water from 72.92 to 26.15 mN m(-1) with CMC of 90 mg l(-1). The predominant rhamnolipid congeners were Rha-C-10-C-10 and Rha-Rha-C-10-C-10 detected by MALDI-TOF MS analysis with approx. 70% relative abundance, although a total of 21 rhamnolipid congeners were accumulated. Increasing the copy number of rhlAB genes efficiently enhanced the production of rhamnolipids by the recombinant P. aeruginosa DAB and thus presents a promising application for the bioremediation process.