Rhodiola rosea contains many active ingredients, such as salidroside and tyrosol. However, separating the active components from Rhodiola rosea crude extract is difficult due to their similar physical properties. Multi-stage centrifugal fractionation extraction method was used due to its strong extraction and separation ability. Three solvents (n-butanol, n-amyl alcohol, and isobutyl acetate) with better distribution coefficient (k) and separation factor (α) were selected from nine organic solvents by single-stage extraction experiment. NaCl was used to break the two-phase emulsification during extraction. A two-step multi-stage extraction process was designed to separate multiple components. The multi-stage fractionation extraction experiments were carried out under the theoretical optimum conditions. In the first step, monomer 6 (tyrosol) was isolated and enriched in organic phase with a purity of over 94% using the above three solvents. In the second step using n-butanol as solvent, monomer 3 (picein) was enriched in organic phase with a purity of 93.6%, and monomer 5 (salidroside) was enriched in aqueous phase with a purity of 85.2%. Finally, the effect of the number of stages was simulated for the separation of picein. The yield of picein reached 80%, and the purity was close to 100% by more than 20 stages.