A highly selective sample cleanup procedure combined with molecularly imprinted SPE was developed for the isolation of crystal violet from seawater and seafood samples. The molecularly imprinted polymer was prepared using crystal violet as the template molecule, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker. The crystal violet-imprinted polymer was used as the selective sorbent for the SPE of crystal violet. An off-line molecularly imprinted SPE method followed by HPLC with diode-array detection for the analysis of crystal violet was also established. Good linearity on the molecularly imprinted SPE columns was obtained from 0 to 200 μg/L (R2 > 0.99). The result demonstrated that the proposed method can be used for the direct determination of crystal violet in seawater and seafood samples. Finally, five samples were analyzed and the following crystal violet concentrations were obtained: 0.92 and 0.52 μg/L in two seawater samples, as well as 0.36 and 0.27 μg/kg in two seafood samples. There is no crystal violet detected in the third seawater sample.