Self‐assembled supramolecular dispersive liquid‐phase microextraction combined with high‐performance liquid chromatography was developed and introduced for simultaneous extraction and determination of the trace level rhein, chrysophanol, and physcion in Rhubarb. Compared with conventional dispersive liquid‐phase microextraction, the proposed method used a self‐assembled ternary supramolecular consisting of the mixed extraction solvent (heptanol and nonanol) and dispersant (acetone) to achieve high enrichment factors of target analytes. Several factors affecting performance were investigated and optimized, including the mixed extraction solvent, type and volume of the dispersant, the pH of sample phase, salt concentration, shaking time, volume of sample phase, centrifugation time, and rate. Meanwhile, the method mechanism of self‐assembled supramolecular dispersive liquid‐phase microextraction was analyzed and described. Under the optimized extraction conditions, the enrichment factors of rhein, chrysophanol, and physcion were 116.5, 325.9, and 356.1, respectively. Good linearities (r ≥ 0.9952) for all analytes, low limits of detection (less than 0.04 ng/mL), satisfactory precisions (0.1–8.9%), and accuracies (recoveries, 88.2–104.1%) were achieved. The experimental results showed that the approach was simple, fast, with short extraction time, high enrichment factors, good linearities, and low limits of detection.