A cinchonine imprinted chiral monolithic column was prepared for the separation of the diastereomers of cinchonine and cinchonidine by in-situ molecular imprinting technique. This type of molecularly imprinted column can be prepared only by a single-step procedure. In order to improve the selectivity and efficiency, a new in-situ molecular imprinting polymerization system was introduced by adopting porogenic solvents of toluene and 1-dodecanol with relatively low polarity and appropriate ratios of polymerization mixture. Diastereomers of cinchonine and cinchonidine were fully separated both under isocratic and gradient elutions on the chiral monolithic column. The broad peak shown in isocratic elution could be improved in gradient elution. Effects of mobile phase composition, flow rate and temperature on retention and separation factor were investigated. Due to the large throughput pores in the chiral monolithic column, low backpressure was observed during the separation process and a separation factor of 3.18 was obtained at 1.0 mL/min. The increase of temperature could improve the separation factor and a maximum separation factor of 5.40 was obtained at 60 degrees C.