Rhamnolipid as a potent natural biosurfactant has a wide range of potential applications, including enhanced oil recovery (EOR), biodegradation, and bioremediation. Rhamnolipid is composed of rhamnose sugar molecule and P-hydroxyalkanoic acid. The rhamnosyltransferase 1 complex (Rh1AB) is the key enzyme responsible for transferring the rhamnose moiety to the P-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid. Through transposome-mediated chromosome integration, the Rh1AB gene was inserted into the chromosome of the Pseudomonas aeruginosa PAO1-rh1A(-) and Escherichia coli BL21 (DE3), neither of which could produce rhamnolipid. After chromosome integration of the Rh1AB gene, the constitute strains P. aeruginosa PEER02 and E. coli TnERAB did produce rhamnolipid. The HPLC/MS spectrum showed that the structure of purified rhamnolipid from P. aeruginosa PEER02 was similar to that from other P. aeruginosa strains, but with different percentage for each of the several congeners. The main congener (near 60%) of purified rhamnolipid from E. coli TnERAB was 3-(3-hydroxydecanoyloxy) decancate (C-10-C-10) with mono-rhamnose. The surfactant performance of rhamnolipid was evaluated by measurement of interfacial tension (IFT) and oil recovery via sand-pack flooding tests. As expected, pH and salt concentration of the rhamnolipid solution significantly affected the IFT properties. With just 250 mg/L rhamnolipid (from P. aeruginosa PEER02 with soybean oil as substrate) in citrate-Na2HPO4, pH 5, 2% NaCl, 42% of oil otherwise trapped was recovered from a sand pack. This result suggests rhamnolipid might be considered for EOR applications.