1, 1'-Bi-2-naphthol is one of the most popular analytes in chiral separation since optically active 1,1'-bi-2-naphthol and its derivatives are frequently used as chiral auxiliaries and ligands in asymmetric synthesis. Therefore, it was employed as template for the preparation of molecularly imprinted polymer (MIP) which was used as liquid chromatographic stationary phase. In this study, the MIP was prepared by bulk polymerization using (R)-(+)-1 , 1'-bi-2-naphthol as template molecule, 4-vinyl pyridine, ethylene glycol dimethacrylate and acetonitrile as functional monomer, cross-linker and porogen respectively. High performance liquid chromatography (HPLC) was employed to study the chiral recognition properties of the resulting MIP. The effect of chromatographic conditions, such as the composition of mobile phase, flow rate and column temperature, on the enantioseparation were systematically investigated. It was experimentally shown that the MIP had specific interactions and high affinity with the template molecule. The MIP could effectively resolve the rac-1, 1'-bi-2-naphthol, and the separation factor was found to be 12.25 under the following conditions: mobile phase, acetonitrile-acetic acid (99: 1, v/v); flow rate, 1.2 mL/min; column temperature, 30 degrees C. The addition of acetic acid to the mobile phase can improve the peak shape, and the lower flow rate and lower column temperature is better for the separation. Further, other substrates which have the similar structure with 1, 1'-bi-2-naphthol, i. e, 5, 6, 7, 8, 5', 6', 7', 8'-octahydro-1, 1'-bi-2-naphthol and 1, 1'-binaphthalenyl-2-amino-2'-ol were also resolved partly by optimizing the HPLC conditions, with separation factors of 1.51 and 2.40, respectively.