Two mutagenesis strains named NY3-1 and NY3-2 were obtained through pseudomonas aeruginosa NY3 inducing with levofloxacin. By comparing the physical and chemical properties of rhamnolipids produced by them, some conclusions were reached as follows: Production, composition and structure characteristics of rhamnolipids produced by mutagenesis strains and wild strains were quite different. Compared with the wild strains，yield of rhamnolipids produced by NY3-1 was improved by 19.57%, while for NY3-2 the yield was decreased by 17.60%. the ratios of amounts of disaccharide and monosaccharide in rhamnolipids produced by NY3、NY3-1、NY3-2 strains were 0.969,0.677 and 1.316 respectively. The infra-red spectrums demonstrated that no C=C unsaturated bond was found in the rhamnolipids produced by NY3 strains, while those produced by NY3-1 and NY3-2 strains contained C=C bond. The results from electrospray ionization mass spectrometry (ESI-MS) analysis showed that all the rhannolipids produced by the three stains contained Rh-C10,while only that produced by two induced strains have high content of Rha-Rha-C10:1 which contain C=C unsaturated bond.