In recent years natural biosurfactants have attracted attention because of their low toxicity, biodegradability, and ecological acceptability. Rhamnolipids is composed of rhamnose sugar molecule and β-hydroxyalkanoic acid. Soil isolates of Pseudomonas fluorescens was used to characterize substrate (Mustard oil, Soybean oil, Olive oil, Palm oil, Sunflower oil and Coconut oil) for highest rhamnolipid production. Highest yield of biosurfactants was obtained from Soybean oil as 7.16 g/L by P. fluorescens. The estimated value of rhamnolipids was 0.437 g/L using Soybean oil from P. fluorescens by Orcinol method. Bacterium was capable of emulsifying a wide range of vegetable oils. The emulsification activity was found stable up to 72 hours. Upon characterizing C:N ratio of higher rhamnolipid was obtained at C:N ratio of 40 (93.75%) for P. fluorescens using Soybean oil as carbon source and Ammonium chloride as nitrogen source. It was revealed that partially purified rhamnolipid of P. fluorescens showed highest emulsification at optimum temperature 4°C for Palm oil, Sunflower oil and Coconut oil (90% all), at pH 6 and pH 7 in Palm oil, Sunflower oil and Coconut oil (90% all) and for 5% NaCl concentration in palm oil (94.44%).