This work focused on the development of an inexpensive carbon source and the improvement of the fermentation-foam fractionation coupling system. The rhamnolipids production capacity of waste frying oil (WFO) was evaluated. The suitable bacterial cultivation of seed liquid and the addition amount of WFO was 16h and 2% (v/v), respectively. A combined strategy of cell immobilization and oil emulsion avoid cell entrainment inside foam and improves the oil mass transfer rate. The immobilization conditions of bacterial cells into alginate-chitosan-alginate (ACA) microcapsules were optimized using the response surface method (RSM). Under the optimal conditions, rhamnolipids production using batch fermentation with immobilized strain reached 7.18 ± 0.23% g/L. WFO was emulsified into a fermentation medium using rhamnolipids as emulsifier (0.5g/L). By monitoring dissolved oxygen, 30mL/min was selected as a suitable air volumetric flow rate for fermentation-foam fractionation coupling operation. The total production and recovery percentage of rhamnolipids were 11.29 ± 0.36g/L and 95.62 ± 0.38%, respectively.