Sophorolipids (SLs) are regarded as one of the most promising biosurfactants. However, high production costs are the main obstacle to extended SLs application. Semi-continuous fermentation, which is based on in-situ separation, is a promising technology for achieving high SLs productivity. In this study, the sedimentation mechanism of SLs was analyzed. The formation of a hydrophobic mixture of SLs and rapeseed oil was a key factor in sedimentation. And the hydrophobicity and density of the mixture determined SLs sedimentation rate. On this basis, ultrasonic enhanced sedimentation technology (UEST) was introduced, by which the sedimentation rates were increased by 46.9% to 485.4% with different ratio of rapeseed oil to SLs. UEST-assisted real-time in-situ separation and semi-continuous fermentation were performed. SLs productivity and yield were 2.15 g/L/h and 0.58 g/g, respectively, simultaneously the loss ratio of cells, glucose, and rapeseed oil were significantly reduced. This study provides the new horizon for optimization of the SLs fermentation process.