Sophorolipids (SLs) are regarded as one of the most promising biosurfactants. They have a low toxicity and are easily degradable without polluting the environment. However, high production costs are the main obstacle to extended SLs application. Semi-continuous fermentation is a promising technology for achieving high SLs productivity, which is based on in-situ separation. In this study, the sedimentation mechanism of SLs was analyzed. The formation of a hydrophobic mixture of SLs and oil was a key factor in sedimentation. The hydrophobicity and density of the mixture determined SLs sedimentation rate. According to the mechanism, ultrasonic enhanced sedimentation technology (UEST) was introduced, by which the sedimentation rates were increased by 46.9% to 485.4% with different Oil/SLs ratios. UEST-assisted real-time rational in-situ separation and semi-continuous fermentation were performed. We observed that SLs productivity and yield were 2.15 g/L and 0.58 g/g, whereas the loss ratio of cells, glucose, and oil was reduced by 68.2%, 16.2%, and 65.5%, respectively. In-situ SLs separation efficiency and rate were increased by 34.5% and 26.4%, respectively. This study provides the foundation and new horizon for the optimization of the SLs fermentation process.