Bacteria could utilize hydrocarbon as a carbon source in an oil contaminated environment. A total of thirty oil contaminated soil samples were collected from the several districts of Tamil nadu for the isolation and characterization of Pseudomonas aeruginosa by using gram staining, motility and biochemical tests, it was compared with standard strain of P.aeruginosa (ATCC 9027). All the Pseudomonas strains isolated from the samples have been reduced the surface tension and showed the emulsification index very effectively. The results of antagonistic test showed, the Pseudomonas strain was effective against Bacillus subtilus and Staphylococcus aureus due to the production of bio surfactant and the genomic DNA of P. aeruginosa was isolated through standard methods and amplified with specific primers (kpd 1 & kpd 2) to ensure the presence of rhl B gene (723 bp- rhamno lipid gene) involved in the production of bio surfactant and the high quality band of amplified gene (723 bp) was observed.