In this paper, the extraction of hyperosides from Hypericum perforatum L. using Cetyl-trimethyl-ammonium bromite (CTAB) reversed micelles was studied and the affecting factors, that is, pH value, CTAB concentration, and existence of anions in the system were comprehensively investigated. The result showed that extraction using CTAB reversed micelles is a very effective method to separate hyperoside from Hypericum perforatum L. This founding is very significant because it demonstrated that extraction using reversed micelles is a promising way to separate and purify materials of small molecules.