Arhamnolipid producing bacterium, Pseudomonas aeruginosa
was previously isolated from Iranian oil over years. Isolated strain was identified by morphological, biochemical, physiological and 16 sr RNA (1). Glycolipid production by isolated bacterium using sugar beet molasses as a carbon and energy source was investigated. Biosurfactant production was quantified by surface tension reduction, Critical Micelle Dilution (CMD), Emulsification Capacity (EC), and Thin Layer Chromatogeraphy. biosurfactants during growth on waste Dates as the primary carbon and nitrogen sources, respectively. After 48 h of growth the culture supernatant fluid had a rhamnose concentration of 0.18 g/L and surface tension was reduced to 20 mN/m ( %).( reduced the interfacial tension against crude oil from 21 mN/m to 0,47 mN/m) Result from the study showed that the growth of the bacteria using molasses as carbon sources is growthassociated. The specific production rate of rhamnolipid with 2 %, 4 %, 6 %, 8 % and 10 % of molasses are 0.00065; 4.556; 8.94; 8.85; and 9.09. respectively The yield of rhamnolipid per biomass with 2%,4%,6%,8% and 10% molasses are 0.003;0.009;0.053;0.041 and 0.213 respectively. The production of rhamnolipid (0.0531 g rhamnolipid/g biomass) is higher compare to the culture grown in aerobic condition (0.04 g rhamnolipid/g biomass) .The rhamnolipids were able to form stable emulsions with n-alkanes, aromatics, crude oil and olive oil. These studies indicate that renewable, relatively inexpensive and easily available resources can be used for important biotechnological processes.