Molecular imprinting is a technique to prepare polymers with predetermined selectivity, specific recognition and predesigned affinity to a desired molecule. The stability and low cost of molecularly imprinted polymers (MIPs) render them attractive for a broad range of applications such as solid-phase extraction, catalysts, bionic sensors, artificial antibodies, and as selective supports in liquid chromatography or CE. Currently, MIP technique has been widely used in chiral separation.In this study, molecularly imprinted polymer is used as chiral stationary phase (CSP) in HPLC and the work mainly consists of condition screening (types of functional monomer & porogen), monolithic stationary phase synthesizing and chromatography condition optimization. In screening part, a series of molecularly imprinted polymers for N-Carbobenzoxy-L-tryptophan (N-Cbz-L-Tryptophan) comprising different functional monomer and porogen were prepared in a small scale to simulate the monolithic stationary phase. By coupling in situ processing and rebinding evaluation, the porous and selective property of the monolith MIPs were investigated. The result shows the cooperation of functional monomer 4-vinylpyridine (4-VP) and porogen 1-dodecanol/toluene can improve MIP?s permeability and chiral selectivity, which is applied for monolithic chiral stationary phase synthesis in following steps. Ten types of CSPs were then prepared in different conditions to investigate the main factors likely to affect the chiral separation. The chromatographic results indicate that polymerization temperature, amount or composition of porogen, template-functional monomer ratio, and mobile phase composition can affect enantiomeric recognition properties of monolithic CSPs. Among these factors, the polymerization temperature can affect the order and stability of template molecule-functional monomer complex in imprinting process; the amount and composition (toluene/1-dodecanol) of porogenic solvent can influence MIP?s permeability, surface area and imprinting efficiency; and template-functional monomer ratio can affect the affinity and amount of selective binding sites. Besides, morphology investigation illustrates that polymerization temperature and porogen can control the pore size distribution through monolithic polymer matrix. Therefore, the producing condition is a compromise of MIP?s imprinting efficiency and permeability.