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Ultrasound and three-phase partitioning (USTPP) were combined to extract and separate polysaccharides (PS) efficiently from Corbicula fluminea crude extracts. The following optimum experimental conditions were established: 20% (w/v) ammonium sulfate concentration, 1:1 (v/v) t-butanol to crude extract ratio, 180W ultrasonic power, 40kHz frequency, 100% duty cycle, 10min irradiation time, and 35°C. The maximum extraction yield of PS obtained from USTPP was approximately 11.22%, which was higher than the maximum yields from conventional three-phase partitioning (TPP; 9.32%) and ultrasound extraction (USE; 6.05%). The extraction time for USTPP was significantly reduced to 10min from 30 and 60min for TPP and USE methods, respectively. The primary chemical structures of PS collected through the three extraction protocols were basically conserved. The synergistic effects of ultrasound and TPP on PS extraction were also determined. Results revealed that USTPP is an effective technique to extract and separate PS from C. fluminea potentially applied in food, cosmetics, and medicines.