A molecularly imprinted polymer solid phase extraction (MISPE) method combined with flow-injection chemiluminescence (FI-CL) for the determination of residual tetracycline (TC) in fish samples is presented. The molecularly imprinted polymer (MIP) of TC was synthesized and particles of this MIP were packed into a polytetrafluoroethylene (PTFE) tube, which was connected into the sampling loop of an eight-way injection valve and served as the MISPE column for on-line selective adsorption of TC. The eluent (CH₃CN : HNO₃ (0.01 mol L⁻¹) = 4 ∶ 1, v ∶ v) was used for extracting the adsorbed TC, which could be detected by its good enhancing effect on the CL reaction between Ce(IV) and rhodamine B. The CL intensity is linear to TC concentration in the range from 4 × 10⁻⁹ to 4 × 10⁻⁷ g mL⁻¹. The detection limit is 1 × 10⁻⁹ g mL⁻¹ (3σ) and the relative standard deviation is 2.4% (n = 9). The conditions of preconcentration, extraction and CL reaction were carefully studied. The selectivity experiment shows that the selectivity and sensitivity of the CL method could be improved greatly when MIP was used as a recognition material in SPE. However, the MISPE column interacted indiscriminately with oxytetracycline (OTC) with a 49 ± 2% binding. An intermediate differential pulsed elution (DPE) step using 3% acetic acid as eluent was employed to remove OTC and other interfering substances. The proposed MISPE-CL method has been applied successfully to the determination of TC in fish samples. At the same time, the binding characteristics of the polymer to tetracycline were evaluated by batch and dynamic methods.