Pure compounds extracted and purified from natural sources are crucial to lead discovery and drug screening. This study presents a novel two-dimensional hyphenation of expanded bed adsorption chromatography (EBAC) and high-speed countercurrent chromatography (HSCCC) for extraction and purification of target compounds from medicinal plants in a single step. The EBAC and HSCCC were hyphenated via a six-port injection valve as an interface. Fractionation of ingredients of Salvia miltiorrhiza and Rhizoma coptidis was performed on the hyphenated system to verify its efficacy. Two compounds were harvested from Salvia miltiorrhiza, one was 52.9 mg of salvianolic acid B with an over 95% purity and the other was 2.1 mg of rosmarinic acid with a 74% purity. Another two components were purified from Rhizoma coptidis, one was 4.6 mg of coptisine with a 98% purity and one was 4.1 mg of berberine with a 82% purity. The processing time was nearly 50% that of the multistep method. The results indicate that the present method is a rapid and green way to harvest targets from medicinal plants in a single step.