A novel gradient elution was efficiently utilized for the separation of the chemical components with a wide range of polarity from the mixed extract of the Chinese medicinal herb Apocynum venetum or mixed standards by high-speed counter-current chromatography. Three sets of solvent systems, n-hexane-ethyl acetate-methanol-water (1.5:3.5:2:4.5 v/v/v/v), ethyl acetate-methanol-water (5:2:5 v/v/v) and n-butanol-methanol-water (5:1:5 v/v/v) were used for the one-step elution. The separation was initiated by filling the column with the lower phase of n-hexane-ethyl acetate-methanol-water (1.5:3.5:2:4.5 v/v/v/v) as a stationary phase followed by elution with the upper phase of n-hexane-ethyl acetate-methanol-water (1.5:3.5:2:4.5 v/v/v/v) to separate the hydrophobic compounds (tail to head). Then the mobile phase was switched to the upper phase of ethyl acetate-methanol-water (5:2:5 v/v/v) to elute the moderate hydrophobic compounds, and finally the hydrophilic compounds still retained in the column were fractionated by eluting the column with the upper phase of n-butanol-methanol-water (5:1:5 v/v/v). A total of 13 compounds including adhyperforin, hyperforin, amentoflavone, biapigenin, quercetin, astragalin, trifolin, isoquercetin, hyperside, acetyled hyperside, rutin, chlorogenic acid and quercetin-3-O-β-D-glucosyl-β-D-glucopyranoside were successfully separated via the three sets of solvent systems in one-step operation for 90 min.