In the past few decades, chemically synthesized surfactants are routinely getting deposited on land and water systems, by industrial and domestic waste. Some of them are toxic to animals, ecosystem, and humans. The demand for microbial surfactants is progressively increasing due to their diverse and heterogeneous nature that can be applied to reduce surface and interfacial tension across a wide range of industrial and environmental applications. Pseudomonas stutzeri, a well-known organism for bioremediation was used for the production of rhamnolipid, along with the use of ultrasound to enhance the productivity of bio-surfactant during the fermentative production. The aim was to identify the sonication regime suitable for improving the productivity of a varied range of bioprocesses and to elucidate the enhancement using response surface methodology. Ultrasound-assisted fermentation resulted in 1.5 folds more rhamnolipid than the conventional method with 150 W ultrasound power, 6 min of irradiation time and 42.5% duty cycle. The maximum yield of rhamnolipid obtained was 4.78 g/L. Various methods for applicability of the produced bio-surfactant were also studied. This study is the first report for ultrasound assisted enhancement of biosurfactant to the best of our knowledge.