Circulating ultrasonic-assisted extraction (CUAE) was developed as a novel method for extraction of medicinal herbs, and the method was validated. In addition, the novel hyphenated technique comprising CUAE coupled with countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC) was developed and applied to the continuous extraction and online isolation of chemical constituents from Ligusticum chuanxiong Hort. The optimum extraction parameters, including the extraction time of 30 min, extraction temperature of 45 °C, ultrasound power of 300 W, and the liquid–solid ratio of 10 mL/g were determined by response surface methodology. Furthermore, a schematic and the mechanism of online CUAE coupled with CCC and CPC were presented. Three lactones, levistolide A (52.2 mg), Z-ligustilide (48.3 mg), and wallichilide (118.2 mg), with respective purities of 95.8, 96.7, and 96.2%, were obtained from 500 g of the L. chuanxiong raw material using CUAE/CCC. In contrast, senkyunolide A (26.2 mg), l...