Molecularly imprinted microspheres (MIMs) were synthesized by micro-suspension polymerization using matrine (MT) as template. The MIMs were employed for solid-phase extraction (SPE) and as chromatographic stationary phase for the determination of MT from the Chinese medicinal plant Sophora flavescens. The effects of the various eluents, their concentrations and volumes on the retention behavior were investigated. The selectivity and capacity of the imprinted microspheres against MT was also discussed. The results showed that the MIMs exhibited stronger specific affinity to MT than to oxymatrine (OMT). Methanol-water (3:7, v/v) was used for washing impurities from the MIMs-SPE cartridge loaded with the herb extracts, while methanol-glacial acetic acid (9:1, v/v) was used for eluting MT. The maximum load of MT and the recovery of MIMs cartridge towards MT were 38.7 microg g(-1) and 71.4%, respectively. The method developed might be used to separate and extract effective constituents from Chinese medicinal plants on a large scale.