Abstract: The imprinted film specific to enrofloxacin was synthesized using polystyrene microtiter plate as support combining surface imprinting technique. A direct competition ELISA was developed with imprinted film as artificial antibody. The results showed that, the detection limit (IC₁₅) and sensitivity (IC₅₀) of the method were 0.80 and 65.93 μg/L, respectively. The specificity of the method was also investigated by comparing the cross-reactivity of other structurally related compounds with that of the template. The developed ELISA showed a low degree of cross-reactivity of these compounds (0%~22.87%) except ciprofloxacin (72.85%), which was the metabolite of enrofloxacin. The established ELISA has been applied in spiked chicken meat and swine urine samples with the recoveries ranging from 97.30% to 103.56% (the adding standard matter amount in chicken meat were 2.5, 15, 75 μg/kg) and 94.48% to 96.53% (the adding standard matter amount in swine urine were 5, 30, 150 μg/kg). The obtained results correlated well to those gained from HPLC method, which demonstrated that the developed ELISA method was reliable for rapid enrofloxacin detection in real samples.