Display omitted •A high rhamnolipids titer (150g/L) was obtained using sequential fed-batch culture.•A high rhamnolipids productivity was well-maintained as 0.4g/Lh for 17 d.•Cell components play major role in this high-performance fermentation strategy.•This process has a great prospect in high-efficiency rhamnolipids production. Rhamnolipids are the most promising biosurfactants, have widespread applications in many fields. However, low yield and productivity in fermentation caused a high production cost and thus prohibited the bulk applications of rhamnolipids in industry. In this study, a sequential fed-batch fermentation process with fill-and-draw operation was developed to improve rhamnolipids production. By utilizing this strategy, the total produced rhamnolipids reached over 150g/L, had a 163% and 102% increase over the traditional batch and fed-batch processes, respectively. This remarkable high production efficiency was achieved by the well-maintained high productivity of 0.4g/Lh for a period of 17 d. Astonishingly, the conversion yield was high as 84%, while this value was only 53.2% and 42.7% in the traditional batch and fed-batch process, respectively. The high-efficiency rhamnolipids production in this sequential fed-batch fermentation could be largely explained by a high presence of cell coupled with the replenishment of nutrients and dilution of toxic byproducts via fill-and-draw operation. In all, this validated fermentation strategy offers a great prospect for high-efficiency production of rhamnolipids in industry.