BIOSURFACTANTS are valuable microbial amphiphilic molecules with effective surface-active and biological properties applicable to several industries and processes. Microbes synthesize them, especially during growth on water-immiscible substrates, providing an alternative to chemically prepared conventional surfactants. The objective of this research was to evaluate factors affecting surfactant production from Pseudomonas aeruginosa which was dependent on some factors including the carbon source, medium composition, pH, temperature and duration of incubation. Use of glucose as a carbon source enhanced the production more than the other carbon sources evaluated. The Plackett-burman statistical design was used to optimize culture conditions and evaluate the most significant variables affecting surfactant production. (NH4)2SO4( 5g/l) and Na2HPO4 (2g/l) were the most significant variables affecting biosurfactant production (emulsification activity) which was increased up to 1.2 fold. The optimum medium composition for biosurfactant production is as follows (g/l) : (NH4)2SO4, 5; KH2PO4 , 5; MgSO4.7H2O , 1.5 ; glucose, 25; Na2HPO4 , 2; Na Cl, 2 and yeast extract , 0.2 0The optimum temperature and pH were 35°C and 6, respectively. Two days of incubation gave the highest production and increased biosurfactant yields up to 1.3 fold. Biosurfactant crude extract showed remarkable stability over a wide range of pH (6-10) and temperature (50-90ºC).