In this work, an on-line SPE-HPLC method with spectrophotometric detection was developed for the determination of coumarins in complex samples. For the on-line cleanup of samples, a molecularly imprinted polymer was packed into the column cartridge and coupled directly with HPLC (MISPE-HPLC) using a column switching system. The separation of coumarins was performed on a C18 core-shell column (100 × 4.6 mm, 5 μm) with a mobile phase consisting of 0.3% acetic acid/acetonitrile with gradient elution at a flow-rate of 1 mL min−1. The total time of the whole analytical run, including the extraction step, was 13.25 min. The on-line MISPE-HPLC method was optimized and validated. The results showed good linearity (0.10–100 μg mL−1) with correlation coefficients higher than 0.99. The LOD values were from 0.03 to 0.15 μg mL−1. The proposed method was successfully applied for analysis of real samples (Cassia cinnamon, chamomile tea, and Tokaj specialty wines) and obtained recoveries varied from 78.7% to 112.2% with an RSD less than 9%.