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Microalgae are new and sustainable sources of starch with higher productivity and flexible production modes than conventional terrestrial crops, but the downstream processes need further development. Here, ultrasonication (with power of 200 W or 300 W and duration of 10, 15, 20, or 25 min) was applied to simultaneously extract and modify starch from a marine microalga Tetraselmis subcordiformis for reducing the digestibility, and an aqueous two-phase system (ATPS) of ethanol/NaH2PO4 was then used to isolate the starches with varied properties. Increasing ultrasonic duration facilitated the partition of starch into the bottom pellet, while enhancing the ultrasonic power was conducive to the allocation in the interphase of the ATPS. The overall starch recovery yield reached 73 ∼ 87 % and showed no significant difference among the ultrasonic conditions tested. The sequential ultrasonication-ATPS process successfully enriched the starch with purities up to 65 % ∼ 88 %, which was among the top levels reported in microalgal starch isolated. Ultrasonication produced more amylose which was mainly fractionated into the interface of the ATPS. The digestibility of the starch was altered under different ultrasonic conditions and varied from different ATPS phases as well, with the one under the ultrasonic power of 200 W for 15 min at the bottom pellet having the highest resistant starch content (RS, 39.7 %). The structural and compositional analysis evidenced that the ultrasonication-ATPS process could exert impacts on the digestibility through altering the surface roughness and fissures of the starch granules, modulating the impurity compositions (protein and lipid) that could interact with starch, and modifying the long- and short-range ordered structures. The developed ultrasonication-ATPS process provided novel insights into the mechanism and strategy for efficient production of functional starch from microalgae with a potential in industrial application.