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A green and effective extraction system based on the use of an ultrasound-assisted ionic liquid aqueous two-phase system (IL-ATPS) was developed for the determination of aconitum alkaloids in lateral roots of Aconitum carmichaeliDebx (Fuzi in Chinese). IL-ATPS parameters, including the type and amount of ionic liquid, amount of salts, and pH value, as well as extraction time, were investigated. Under the optimized conditions, the recoveries of the analytes varied from 80.37 to 107.18%. The detection limits and quantification limits for the analytes were achieved within the ranges of 0.048–0.082 μg g−1and 0.16–0.30 μg g−1, respectively. After extraction, the recovery of ionic liquids without the use of organic solvents was also confirmed. The hydrophilic ionic liquids (1-hexyl-3-methylimidazolium bromide) were converted into hydrophobic ionic liquids (1-hexyl-3-methylimidazolium hexafluorophosphate) by a metathesis reaction, and in situliquid–liquid microextraction was triggered simultaneously. Finally, 0.1 mol L−1HCl aqueous solution (containing 5% Tween-20) was used as the back-extraction agent to recover the ionic liquids.