Abstract In this work, we prepared a monolithic and surface initiated molecularly imprinted polymeric (MIP) column for HPLC and explored its application for template separation from plant extract. The silica beads (40–60 μm) were coupled with initiator on the surface and then packed in to a stainless steel HPLC column. The pre-polymerization mixture (the template, functional monomer and crosslinker were emodin, acrylamide and divinylbenzene, respectively) was injected into the column and polymerized by thermal initiation. The prepared MIP column exhibited excellent retention capability and large imprinted factor for template (the retention time and imprinted factor for emodin on MIP column were 16.26 min and 7.21, respectively). Moreover, the emodin-molecularly imprinted polymeric column could be applied to separate emodin from alcohol extract of Rheum palmatum L. at semi-preparative scale and 1.2 mg of emodin was obtained in 4 h.