A high affinity polyclonal antibody-based enzyme linked immunosorbent assay (ELISA) was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1:5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 μg/ml, and the detection limit was 0.02 μg/ml for the assay. The overall recoveries and the coefficients of variation (CVs) were in the range of 82% ～127% and 3.5%～8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol (ranging from 0.2 to 10 μg/ml) were detected by the ELISA and liquid chromatography (LC) method, and good correlations were obtained between the two methods (R2=0.9643). We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an alternative for the conventional LC method for zeranol in bovine urine.