ABSTRACT: Biosurfactants are products of a wide range of microorganisms which have high level of surface activity. These are amphilye molecules constituted of hydrophobic and hydrophilic parts in this study, microorganisms separated from IRAN oil wells were used for biosurfactant production. After isolation of this organisems, its type was determined as pseudomonas aeruginosa. Main object of this study is optimization of pseudomonas aeruginosa growth condition to produce rhamnolipid. To do this, a mineral salts media is used and many different factors were studied. In an experiment to determine the best culture media for production of rhamnolipid, it was shown that between different mineral salts of 1M, 2M, 3M and 4M, the best culture media is 3M which in it, glucose is applied as carbon source. In rhamnolipid production is different incubation times intervals and conditions, it was shown that at fixed conditions of 2% inoculation, C/N=16, aeration=200 rpm, pH=6.8, T=30°C and 3M media, maximum production obtained after 96 hrs. After 24 hrs. rhamnose production is 0.06 g/l, at 48 hrs is 0.1, at 72 hrs is 0.17 at 96 hrs is 0.22, at 120 hrs is 0.15 g/l. In our experiments, maximum emulsification of crude oil in the condition of C/N=28, inoculation=2%, aeration=200 rpm, pH=7.5 and T=35°C has been 82.5 %.