Sophorolipids are environmentally friendly biosurfactants and hence their demand has greatly increased during last decade, especially in cleaning and cosmetic applications. However, their higher production cost becomes a major barrier for wider applications compared to their counterparts Le. petrol-derived surfactants. In this investigation, a novel and efficient technology for the in situ separation of sophorolipids has been proposed, including froth flotation followed by recovery of entrained cells through washing. First, independent cascade separators having a pre-separator and a froth separator were designed. 75% of sophorolipids were separated in the froth separator within 12 min after adjusting oil to sophorolipids ratio (0.08-0.012) in the bioreactor. Washing was then applied to recover over 90% of entrained cells in the froth. Finally, using 5 L bioreactor three rounds of in situ separation were carried out to achieve a final production of 342 g/L (normalized to initial volume) for 221 h, an improvement of 41% compared to fed-batch fermentation. Besides, the overall productivity reached 1.55 g/L/h. Overall, this investigation provides an in situ separation technology with controllable frothing and is applicable to an industrial scale. Also it maximizes the production potential of strain along with lowering the production cost of sophorolipids significantly.