ABSTRACT:                 In the last decades, biosurfactants have attracted attention of many scientists because of their low toxicity, biodegradability and ecological acceptability. Nevertheless, their use is extremely limited due to the high cost when compared with chemical surfactants. In this study we have isolated bacteria from different environmental sources located in Sarris field (Zawia–Libya). The isolate was identified as Pseudomonas aeruginosa by comparing its 16S rDNA sequence with those available in The National Center for Biotechnology Information (NCBI) gene bank (accession number was AJ746 135). Using gel electrophoresis technique, the length of DNA was identified. The strain was selected for its ability to produce extra cellular products (biosurfactants) able to reduce surface tension and emulsify the kerosene. The production of rhamnolipid was carried out using a well-controlled one-liter laboratory-scale flask fermentor. In the same time, optimum concentration of substrate (glycerol) was investigated and phenol sulphuric acid test was conducted to measure the concentration of produced rhamnolipid. The identification tests such as specific growth rate (µ), protein measurement, emulsification activity and stability test were also studied. The results showed that the extracellular surface active agent produced by the indigenous strain P. aeruginosa was identified as Rhamnolipid, which is one of the most commonly used biosurfactants with the ability to reduce surface tension of water. The maximum concentration of rhamnolipid isolated using 1% glycerol which was 170mg/l. This biosurfactant had excellent emulsification index (E24) of 70% and stability of 67% comparable to or better than that from the conventional rhamnolipid which was 66% and 58% respectively.