Abstract: Chlortetracycline hydrochloride can enhane the chemiluminescence of Ce(Ⅳ) and Rh6G in acidic medium. So novel molecularly imprinted polymer solid-phase extraction(MISPE) method combined with flow-injection chemiluminescence for determining chlortetracycline hydrochloride was developed. The MIP was synthesized by using tetracycline as the imprinting molecule which has a structure similar to that of chlortetracycline. A polytetrafluoroe-thylene(PTFE) tube packed with the particles of MIP was employed as an MISPE micro-column and was connected to the sampling loop of the eight-way injection valve for the preconcentration and extraction of chlortetracycline hydrochloride. The eluent was used as the carrier for extracting the adsorbed chlortetracycline hydrochloride to react with a mixture of cerium(Ⅳ) and rhodamine6G in the flow cell to produce strong CL. Intensity of CL is linearly changed with the concentration of chlortetracycline hydrochloride from 2×10^-8 g/mL to 1×10^-6 g/mL and the limit detection is 4×10^-9 g/mL(3σ). This method has been successfully applied to the determination of chlortetracycline hydrochloride in liver of chicken sample.