A time-resolved fluoroimmunoassay (TRFIA) technique is developed to detect enrofloxacin (ENR) contamination in food. By using ENR-ovalbumin, anti-ENR antibodies and europium-labeled goat anti-rabbit antibodies, we establish an indirect and competitive method for ENR-TRFIA. The sensitivity of the method is high, with a detection limit of 0.01 ng/mL. The tests show that the technique's sensitivity is 1 μg/kg in eel, pork and chicken, and 1 μg/L in honey. The detection range attained is 0.01-100 ng/mL and within the detection range the intra- and inter-batch coefficients of variation of the ENR-TRFIA method are 2.4% and 9.2%, respectively. The data obtained from eel samples by TRFIA and enzyme-linked immunoassay are in good agreement. The assay did not cross-react with other quinolones, which commonly exist in food. The study suggests that ENR-TRFIA is a simple, sensitive and economic method of screening large quantities of samples, and has good prospects for application.